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The Journal of Immunology, 2006, 177: 7173-7183.
Copyright © 2006 by The American Association of Immunologists, Inc.

IL-1β and TNF-{alpha} Regulation of the Adenosine Receptor (A2A) Expression: Differential Requirement for NF-{kappa}B Binding to the Proximal Promoter1

Silvana Morello*,{dagger}, Kazuhiro Ito*, Satoshi Yamamura*, Kang-Yun Lee*, Elen Jazrawi*, Patricia DeSouza*, Peter Barnes*, Carla Cicala{dagger} and Ian M. Adcock2,*

* Airways Disease Section, NHLI Imperial College London, London, United Kingdom; and {dagger} Department of Experimental Pharmacology, University of Naples "FedericoII," Naples, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Adenosine is a potent endogenous regulator of airway inflammation that acts through specific receptor subtypes that can either cause constriction (A1R, A2BR, and A3R) or relaxation (A2AR) of the airways. We therefore examined the effects of key inflammatory mediators on the expression of the A2AR in a lung epithelial cell line (A549). IL-1β and TNF-{alpha} increased the expression of the A2AR gene at the mRNA and protein levels. In contrast, LPS had no effect on A2AR gene expression. IL-1β and TNF-{alpha} rapidly activated p50 and p65, but not C-Rel, RelB, or p52, and both IL-1β- and TNF-{alpha}-stimulated A2AR expression was inhibited by the I{kappa}B kinase 2 inhibitor AS602868 in a concentration-dependent manner. Using chromatin immunoprecipitation assays, we demonstrate that IL-1β can enhance p65 association with putative {kappa}B binding sites in the A2AR promoter in a temporal manner. In contrast, TNF-{alpha} failed to enhance p65 binding to these putative sites. Functionally, the two most 5' {kappa}B sites were important for IL-1β-, but not TNF-{alpha}-, induced A2AR promoter reporter gene activity. Finally, neither TNF-{alpha} nor Il-1β had any effect on A2AR mRNA transcript degradation. These results directly implicate a major role for NF-{kappa}B in the regulation of A2AR gene transcription by IL-1β and TNF-{alpha} but suggest that the effects of TNF-{alpha} on A2AR gene transcription are not mediated through the proximal promoter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Adenosine is a potent endogenous regulator of inflammation that exerts its biological actions through binding to specific G protein-coupled receptors on target cells (1, 2). The expression of these receptors is widely distributed among tissues, and this signaling pathway has been implicated to exert important effects in many physiological systems (3, 4), in cell growth and proliferation (5), and in apoptosis (6, 7). Adenosine clearly plays a role in inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (COPD)3 (8, 9). It is known that administration of adenosine by inhalation causes concentration-related bronchoconstriction in subjects with asthma and COPD (10, 11) but not in normal volunteers. Adenosine is able to influence the function of cells involved in the exacerbation of asthma, including mast cells (4), macrophages (12, 13), neutrophils (9), lymphocytes (14), eosinophils (15), smooth muscle cells (16), and epithelial cells (17). In addition to its putative role on inflammatory and immune responses, adenosine also appears to exert important effects on transepithelial electrolyte secretion in human airways. Adenosine receptor activation has been shown to activate transmembrane conductance regulator (cystic fibrosis transmembrane conductance regulator) Cl– channels (18), non-cystic fibrosis transmembrane conductance regulator Cl– channels (19, 20), amiloride-sensitive Na+ channels (21, 22), and Ca2+-dependent K+ channels (23).

Adenosine can act through any of four distinct G protein-coupled receptors, and various studies have implicated three of these four receptors (A1R, A2BR, and A3R) in adenosine- induced bronchoconstriction. Conversely, the stimulation of A2AR leads to bronchodilation by inhibiting the release of mediators of the immune response from mast cells (24, 25, 26). The expression and the function of adenosine receptors are regulated by endogenous factors involved in the inflammatory response such as growth factors (27, 28), glucocorticoids (29, 30), and other cytokines (31, 32). For example, it has been demonstrated that A3R are up-regulated in activated human lymphocytes (33); inflammatory cytokines up-regulate the function and expression of A2AR in human monocytic THP-1 cells (31) and in rat PC12 cells (32); nerve growth factor down-regulates A2AR expression in PC12 cells (27, 34) and IFN-{gamma} up-regulates A2BR expression in macrophages (35). We have recently shown that the expression of the A2BR is increased in the airways and lungs of smokers with COPD (36) which is characterized by an increased inflammatory response involving IL-1β and TNF-{alpha} (37).

Epithelial cells not only form a physical barrier between the airways and the environment but are important inflammatory cells that respond initially to most airborne insults and are a target for inhaled drugs (38). Recently, it has been demonstrated that human epithelial cells express mRNA for A1, A2A, and A2B receptors (39). However, the expression of A2A receptor and its regulation under inflammatory conditions in epithelial cells have not been documented. In this study, we investigated the mechanisms by which the proinflammatory stimuli IL-1β and TNF-{alpha} enhance A2AR gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

Normal human tracheobronchial epithelial cells were obtained from PeproTech and cultured exactly as described by the supplier. Cells were cultured in bronchial epithelial growth medium supplemented with bovine pituitary extract (50 µg/ml), hydrocortisone (0.5 µg/ml), human epidermal growth factor (0.5 ng/ml), epinephrine (0.5 µg/ml), transferrin (10 µg/ml), insulin (5 µg/ml), all-trans retinoic acid (0.1 ng/ml), triiodothyronine (6.5 ng/ml), gentamicin (50 µg/ml), amphotericin B (50 ng/ml), and BSA (1.5 µg/ml) and were used at passages two to three at 70% confluency. A549 cells, a human lung adenoma cell line (CCL185), were grown in DMEM containing 10% FCS before incubation for 48 h in serum-free medium. At the onset of each experiment, cells were placed in fresh medium and then cultured in the presence of IL-1β (0.1–1-10 ng/ml), TNF-{alpha} (1 to10–50 ng/ml), LPS (10–50 µg/ml; Salmonella enteteridis), or other reagents followed by further analyses. Recombinant human IL-1β and recombinant human TNF-{alpha} were purchased from R&D Systems. SP600125 (5 µM; Calbiochem) and SB203580 (1 µM; Sigma-Aldrich) were used to inhibit the JNK and p38 MAPK pathways. The I{kappa}B kinase 2 (IKK2) inhibitor (AS602868) was provided by Dr. M. Dreano (Serono). The A2AR agonist CSG21680 was obtained from Sigma-Aldrich. Cells were treated with SP600125, SB203580, AS602868, or the vehicle (DMSO), for 0.5 h before stimulation with IL-1β or TNF-{alpha} for 2–24 h. In some experiments, A549 cells were treated with IL-1β (1 ng/ml) or TNF-{alpha} (10 ng/ml) for 24 h before restimulation with IL-1β or TNF-{alpha}. After 6 h, the cell supernatants were analyzed for GM-CSF and secretary leukocyte protease inhibitor (SLPI)release by ELISA (R&D Systems).

Construction of reporter plasmids

Each fragment of human adenosine A2AR promoter was generated by PCR using Platinum Pfx DNA Polymerase (Invitrogen Life Technologies) with human genomic DNA as a template. These fragments were cloned into pGL3 basic vector (Promega). There are five putative NF-{kappa}B binding sites in the promoter region. We constructed five different length reporter plasmids using PCR and restriction enzymes. The –339/–1 fragment was cloned by PCR using an NheI site (underlined) within the forward primer (5'-ACGCGTGCTAGCCCAAGTGTGGGGTAAGG G-3') and an NcoI site (underlined) within the reverse primer (5'-A AGACACCATGGCCACAGACGACAGGC-3'). The PCR product was digested with NheI and NcoI and cloned into the NheI/NcoI-digested pGL3 basic vector. –1263/–1, –2301/–1 and –2747/–1 fragments were cloned using a two-step PCR protocol. Each upstream fragment was cloned using the NheI site (underlined) within the forward primer (5'-ACGCGTGCTAGCGCGTGGGAGGAGGGAGGT-3' for –1263/–1 fragment, 5'-ACGCGTGCTAGCCCGACAGCATTGGAGTTG-3' for –1979/–1, 5'-ACGCGTGCTAGCGTGTTCCTTGAGTGTGGC-3' for –2301/-1, 5'-ACGCGTGCTAGCGGCTCTGGGT GATGGG AC-3' for –2747/–1) and reverse primer (5'-AGGGCTTTTTCACAGAGGACTCTGAGTCTC-3') with human genomic DNA as a template. The downstream fragment was cloned using the forward primer (5'-AAGTGTGGGGTAAGGGGTGGCACTTTCCGC-3') and the reverse primer (5'-TAGAATGGCGCCGGGCCTTTC-3') with the –339/–1 pGL3 basic plasmid as the template. Each fragment was cloned using the same forward and reverse primers (5'-TAG AATGGCGCCGGGCCTTTC-3'). The PCR product was digested with NheI and NarI and cloned into the NheI/NarI-digested pGL3 basic vector. The –2932/–1 fragment was cloned using forward (5'-ACGCGTGCTACGCCCGCC AG-3') and reverse primers (5'-GCTGGCCCCGGGCTGCCTCCCACCC-3') with human gnomic DNA as a template. The PCR product was digested with NheI and XmaI and cloned into the NheI/XmaI-digested –2301/–1 pGL3 plasmid.

RNA isolation and real-time quantitative PCR

RNA extraction from A549 cells was performed using an RNeasy Mini kit according to the manufacturer’s instructions (Qiagen). Sample RNA was quantified by spectrophotometry and the complementary DNA was synthesized from total RNA as described previously (40). Thermal cycling and SYBR Green fluorescence detection was performed in a Real-Time PCR LightCycler System (Rotor-Gene 3000 Sequence Detection System; Corbett Research). Sample and control mRNAs were quantified relative to an internal reference RNA, β-actin (Ambion). Thermal cycling conditions were 15 min at 95°C, followed by 45 cycles of 15 s at 94°C, 25 s at 60°C, 25 s at 72°C, and 5 s at 86°C. A melting curve analysis was added after the final PCR cycle to evaluate the presence of nonspecific PCR products and primer dimers. A nontemplate control was run with every assay, and all determinations were performed at least in duplicate to achieve reproducibility. Primer pairs of A2AR were as follow: forward 5-AGGCAGCAAGAACCTTTCAA-3 and reverse 5-CTAAGGAGCTCCACGTCTGG-3. In conventional RT-PCR, all primers generated only one amplification band resolved by agarose gel electrophoresis and were visualized with ethidium bromide, demonstrating specificity.

Western blotting analysis

Immunoblot analysis was performed according to Laemmli (41). Briefly, 40 µg of total whole-cell protein per lane was separated through 10% denaturing polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in PBS-Tween 20 (0.1% v/v) and then incubated overnight at 4°C with goat anti-A2AR polyclonal Ab (1/500; Santa Cruz Biotechnology). The secondary Ab was HRP-conjugated sheep anti-goat (diluted 1/4000) and ECL reagent (Amersham Pharmacia Biotech.) was used for detection. Each filter was reprobed with mouse anti-human β-actin mAb (Abcam). The bands, which were visualized by autoradiography, were quantified using a densitometer with GRAB-IT and GELWORKS software (Ultraviolet Products).

Nuclear extract preparation and NF-{kappa}B and AP-1 activation assays

Nuclear extracts from A549 cells were prepared using the Nuclear Extraction Kit from Active Motif. NF-{kappa}B subunit activation was measured with a TransAM NF-{kappa}B Family kit (Active Motif) according to the manufacturer’s instructions.

Chromatin immunoprecipitation (ChIP) assay

After stimulation, cells were fixed by adding formaldehyde directly to the medium to a final concentration of 1% and treated as previously described (42). Cells were resuspended in 200 µl of SDS lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris-HCl (pH 8.1); complete proteinase inhibitors mixture). Chromatin was shared by sonication (6 x 10-s pulses) on ice, centrifuged to pellet debris, and diluted 10 times in dilution buffer (167 mM NaCl, 16.7 mM Tris-HCl, 1.2 mM EDTA, 1.1% Triton X-100, and 0.01% SDS). Extracts were precleared for 30 min at 4°C with 80 µl of a 50% suspension of salmon sperm DNA-agarose A slurry (Upstate Biotechnology). Immunoprecipitations were conducted at 4°C overnight. Immunocomplexes were collected with salmon sperm DNA-agarose A slurry and washed with LiCl wash buffer and Tris-EDTA. Immune complexes were eluted by adding elution buffer (1% SDS and 0.1 M NaHCO3), and protein-DNA cross-links were reverted by heating at 65°C for 4 h. After proteinase K digestion (70 µg/ml, 1 h at 45°C), DNA was extracted by phenol-chloroform, precipitated with ethanol, 0.3 M NaHCOOH, 20 µg of glycerol, and resuspended in 50 µl of Tris-EDTA. Quantitative real-time PCR analysis was used to determine the percentage of A2AR promoter input DNA that was bound by p65. Sequences of promoter-specific primers are given below: A2AR I forward (5'-GCAGGAAGGTGGCTTCAGTA-3') and A2AR I reverse (5'-CCTGGGTACCTGCCTGTGTA-3'); A2AR II forward (5'-GTCAGGCTTCTCCCAAACTG-3') and A2AR II reverse (5'-TGCCAAAGTGAAGGAGGACT-3'); A2AR III forward (5'-GCCAAGAGCATCAGGGATAA-3') and A2AR III reverse (5'-GCCATTCACAAAGTGTGTCG-3'); A2AR IV forward (5'-GTCAGCCAGGCAGAGGAG-3') and A2AR IV reverse (5'-CGCTGCAGCTGACTGTGA-3'); A2AR V forward (5'-GGGTAGGGTGGGATCTGAAA-3') and A2AR V reverse (5'-CCGATGAGAAGCTGAACCAT-3'); GM-CSF forward (5'-CTGACCACCTAGGGAAAAGGC-3') and GM-CSF reverse (5'-CAGCCACATCCTCCAGAGAAC-3').

Measurement of p38 MAPK and JNK activity

JNK and p38 MAPK activity following IL-1β and TNF-{alpha} stimulation was determined by a fast activated cell-based ELISA (FACE) assay (Active Motif) according to the manufacturer’s instructions.

Transfection and reporter assay

For transient transfection, cells were trypsinized, plated in 24-well plates in DMEM plus 10% FBS (7.5 x 104 cells/well), and incubated overnight. The next day, cells were transfected with 2 µl/well LipofectAMINE 2000 with 0.7 µg of reporter plasmid and 0.3 µg of β-galactosidase plasmid. The following day, cells were stimulated with IL-1β (1 ng/ml) or TNF-{alpha} (10 ng/ml) and incubated for 6 h. The cells were washed in PBS and lysed in 200 µl of lysis buffer on ice. Luciferase activity was measured in a TD-20/20 luminometer (Turner Designs) using 2 µl of cell lysate. Luciferase activity was normalized to β-galactosidase activity.

Statistical analysis

Results are expressed as mean ± SEM and the one-way ANOVA test was used for statistical analysis followed by Bonferroni’s multiple comparison test. A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Induction of A2AR gene expression by proinflammatory cytokines

In primary human airway epithelial cells, both IL-1β and TNF-{alpha} induce the expression of adenosine A2AR mRNA expression by 3- to 4-fold after 6 h (Fig. 1). This effect was attenuated by the NF-{kappa}B inhibitor AS602868 (1 µM; Fig. 1). We observed a concentration-dependent increase in the expression of A2AR RNA within 2 h of stimulation with IL-1β (~3- to 4-fold) and this increase was enhanced (~8-fold) at 6 h (Fig. 2a) before declining over the next 18 h in A549 cells. Increases in A2AR mRNA expression were observed from 0.1 to 10 ng/ml IL-1β, with a peak at 10 ng/ml, and this concentration point was used for subsequent experiments. Similar results were obtained when cells were stimulated with TNF-{alpha}. As shown in Fig. 2b, a significant increase in A2AR mRNA expression was seen by 2 h after TNF-{alpha} stimulation, reaching the maximum value at 6 h at a 10 ng/ml concentration. In contrast, no increase in A2AR mRNA was observed after LPS stimulation (data not shown) at concentrations of 10 and 50 µg/ml.


Figure 1
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  		FIGURE 1. Effect of IL-1β (1 ng/ml) and TNF-{alpha} (10 ng/ml) on A2AR mRNA expression in primary human airway epithelial cells after 6 h of stimulation. Induction was prevented by pretreatment of cells with the IKK2 inhibitor AS602868 (5 µM). Total RNA was extracted and analyzed by quantitative real-time PCR and compared with expression of β-actin mRNA. Data represent mean ± SEM of the ratio of A2AR:β-actin mRNA; *, p < 0.05 compared with control untreated cells; #, p < 0.05 compared with stimulated cells, n = 3.

 

Figure 2
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  		FIGURE 2. Concentration- and time-dependent increase of A2AR mRNA expression. A549 cells were treated with IL-1β (a; 0.1, 1, and 10 ng/ml) or TNF-{alpha} (b; 1, 10, and 50 ng/ml) for various time intervals up to 24 h. Total RNA was extracted and analyzed by quantitative real-time PCR. Data represent mean ± SEM fold increase, n = 5 for a and 4–7 for each point in b.

 
Western blot analysis was performed after 24 h to determine whether the increased A2AR mRNA was associated with increased expression of A2AR protein. A2AR protein level was increased by both TNF-{alpha} and IL-1β (Fig. 3a). In particular, IL-1β induced a significant increase in A2AR protein expression at both 1 and 10 ng/ml (Fig. 3b) while TNF-{alpha} induced a concentration-dependent increase in A2AR protein level (Fig. 3c). This suggested that both IL-1β- and TNF-{alpha}-induced A2AR protein production was related, at least in part, to the increased expression of A2AR mRNA. The reduced induction of adenosine A2A receptor protein expression at 24 h (2-fold) by both IL-1β and TNF-{alpha} compared with the greater effects on adenosine A2AR mRNA expression may be a temporal effect in that prolonged incubation of up to 48–72 h may allow a greater induction of protein.


Figure 3
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  		FIGURE 3. Quantitation of A2AR protein expression after 24 h in IL-1β- (a) and TNF-{alpha}-stimulated (b) A549 cells performed by densitometric analysis of Western blots. Data are expressed as mean ± SEM percentage of control. *, p < 0.05 and **, p < 0.01 vs control ({square}), n = 3 for each experiment.

 
Induction of A2AR gene expression by IL-1β is NF-{kappa}B dependent

To evaluate the possible involvement of NF-{kappa}B in the up-regulation of A2AR mRNA in response to IL-1β and TNF-{alpha}, cells were treated with a selective IKK2 inhibitor (AS602868) 0.5 h before stimulation with IL-1β (10 ng/ml) or TNF-{alpha} (10 ng/ml). As shown in Fig. 4, AS602868 abrogated the increase in A2AR RNA expression induced by IL-1β and TNF-{alpha} in a concentration-dependent manner with no significant difference between the IC50 for either stimulus (0.55 ± 0.06 vs 1.05 ± 0.26, p > 0.05, n = 3.6). In addition, although the effect of AS602868 was significant at 1 µM for IL-1β-stimulated, but not TNF-{alpha}-stimulated, A2AR expression, this was not significantly different between groups (p > 0.1). Administration of AS602868 alone had no effect on the expression of A2AR.


Figure 4
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  		FIGURE 4. Effect of the NF-{kappa}B inhibitor AS602868 on the A2AR mRNA level in cells stimulated with IL-1β (a; 10 ng/ml) or TNF-{alpha} (b; 10 ng/ml). AS602868 (1, 5, and 10 µM) was added to the cells 0.5 h before cytokine treatment and cultured for 2 h. Results are expressed as mean ± SEM; *, p < 0.05; **, p < 0.01; and ***, p < 0.001 vs cytokine-treated cells, n = 4–8 for each point.

 
Activation of NF-{kappa}B subunits by IL-1β and TNF-{alpha}

We investigated the induction of NF-{kappa}B family members following IL-1β and TNF-{alpha} stimulation. IL-1β induced a significant induction of both p65 and p50 subunits within 10 min that was maintained for 4 h in the case of p65, with a peak activation at 60 min (Fig. 5). Activation of p50 continued to increase over the 4-h time course of the experiment. In contrast, there is no significant induction of C-Rel activation following IL-1β stimulation and p52 and RelB activity is only increased at 3–4 h, by which time significant activation of A2AR mRNA expression has already occurred (Fig. 2).


Figure 5
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  		FIGURE 5. Time course of IL-1β induction of NF-{kappa}B subunit activation. Cells were stimulated with IL-1β (10 ng/ml) for various time points up to 4 h and the DNA-binding activity of p65 (a), p50 (b), C-Rel (c), p52 (d), and RelB (e) was determined by a TransAm kit. Results are expressed as mean ± SEM; **, p < 0.01 and ***, p < 0.001 vs value at t = 0. n = 3 at all points for all subunits except for p65, where n = 5.

 
TNF-{alpha} also caused a marked activation of p65 and p50 across all time points studied (Fig. 6). As with IL-1β, there was no activation of C-Rel in these cells following stimulation for up to 4 h. Activation of p52 followed a similar time course to that induced by IL-1β being activated at 3–4 h after stimulation. However, TNF-{alpha} induced a small (2-fold) but significant activation of RelB within 30 min that was further increased after 3–4 h.


Figure 6
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  		FIGURE 6. Time course of TNF-{alpha} induction of NF-{kappa}B subunit activation. Cells were stimulated with TNF-{alpha} (10 ng/ml) for various time points up to 4 h, and the DNA-binding activity of p65 (a), p50 (b), C-Rel (c), p52 (d), and RelB (e) was determined by a TransAm kit. Results are expressed as mean ± SEM; *, p < 0.05; **, p < 0.01; and ***, p < 0.001 vs value at t = 0. n = 3 at all points for all subunits except for p65, where n = 5.

 
Kinetic analysis of NF-{kappa}B recruitment to the A2AR promoter

Analysis of the human A2AR promoter sequence (AP000355 Homo sapiens genomic DNA, chromosome 22q11.2) using Transfac (43, 44) indicates the presence of five putative NF-{kappa}B binding sites upstream of the transcriptional start that are closely related to the sequence of the NF-{kappa}B consensus site and to other well-characterized NF-{kappa}B sites (45) (Table I). ChIP assays were used to investigate the temporal binding of p65 NF-{kappa}B to the individual putative NF-{kappa}B sites within the native A2AR promoter following IL-1β and TNF-{alpha} stimulation. As a positive control, we initially analyzed p65 recruitment to the GM-CSF promoter, a canonical NF-{kappa}B-dependent gene, following IL-1β treatment. A rapid induction of p65 GM-CSF promoter association was observed which peaked at 20 min before returning to control levels within 2–4 h (Fig. 7a). Enrichment of promoter sequences was not detected when the Ab was omitted from the immunoprecipitation reaction, confirming the specificity of the ChIP assay (data not shown). Similarly, recruitment of p65 to each of the putative NF-{kappa}B sites in the A2AR promoter was extremely rapid (Fig. 7b). In contrast, there was no specific recruitment of p65 to any of the putative NF-{kappa}B sites in the A2AR promoter following TNF-{alpha} stimulation at any time point studied (Fig. 8), suggesting that TNF-{alpha}-induced A2AR mRNA expression is independent of NF-{kappa}B promoter binding.


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  		Table I. NF-{kappa}B sites in the A2AR promoter/enhancer

 

Figure 7
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  		FIGURE 7. Early and transient recruitment of NF-{kappa}B to the {kappa}B site in the GM-CSF promoter (a) and to the five potential {kappa}B sites in the A2AR promoter (b). A549 cells were treated with IL-1β (10 ng/ml), and ChIP assays were performed with an anti-p65 affinity-purified rabbit polyclonal Ab. p65-precipitated DNA was analyzed by quantitative real-time PCR with promoter-specific primers amplifying the A2AR promoter or GM-CSF promoter. The results are expressed as mean ± SEM, n = 3–4 for each time point.

 

Figure 8
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  		FIGURE 8. No recruitment of NF-{kappa}B (p65) to the five potential {kappa}B sites in the A2AR promoter following TNF-{alpha} (10 ng/ml) stimulation. A549 cells were treated with TNF-{alpha}, and ChIP assays were performed with an anti-p65 affinity-purified rabbit polyclonal Ab. p65-precipitated DNA was analyzed by quantitative real-time PCR with promoter-specific primers amplifying the A2AR promoter. The results are expressed as mean ± SEM, n = 4–5 for each time point.

 
Effect of sequential deletion of {kappa}B sites on IL-1β- and TNF-{alpha}-induced A2AR promoter-luciferase activity

IL-1β induced a significant (p < 0.01) increase in A2AR-luciferase activity and sequential deletion of {kappa}B sites resulted in complete loss of induction following deletion of site II (Fig. 9). Deletion of site IV restored 50% of the activity with IL-1β implicating a suppressor role for this region. Further deletion of site V again resulted in no IL-1β-induced luciferase activity. In contrast, TNF-{alpha} was unable to increase A2AR promoter-luciferase activity above baseline levels and sequential deletion of {kappa}B sites did not affect reporter gene activity (Fig. 9).


Figure 9
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  		FIGURE 9. Effect of sequential deletion of {kappa}B sites on IL-1β and TNF-{alpha}-induced A2AR promoter-luciferase activity. Cells were transfected with A2AR-luciferase and β-galactosidase expression plasmids, and the relative IL-1β- and TNF-{alpha}-stimulated activity was measured. IL-1β-stimulated activity was taken as 100% and the effect of {kappa}B site deletion was examined. The results are expressed as mean ± SEM of at least three independent observations.

 
JNK and p38 MAPK inhibitors

The expression of A2AR mRNA was also under the control of the proinflammatory JNK and p38 MAPK pathways. Inhibition of JNK (SP600125) and p38 MAPK (SB203580) significantly reduced both IL-1β - and TNF-{alpha}-stimulated p38 MAPK (Fig. 10a) and c-Jun (Fig. 10b) phosphorylation as detected by FACE assays. Inhibition of these pathways subsequently attenuated induction of A2AR mRNA expression (Fig. 10, c and d), suggesting that differential requirement for these pathways does not account for the differences in A2AR induction by IL-1β and TNF-{alpha}.


Figure 10
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  		FIGURE 10. Effect of p38 MAPK inhibitor (SB203580) and JNK inhibitor (SP600125) on A2AR mRNA level induced by IL-1β (10 ng/ml) or TNF-{alpha} (10 ng/ml). Cells were treated with IL-1β or TNF-{alpha} for 10 min (a) or 30 min (b) and the activation of p38 MAPK (a) and c-Jun (b) was detected by FACE assay in the presence or absence of SB203580 (3 µM) or SP600125 (3 µM). Cells were subsequently treated with IL-1β or TNF-{alpha} for 2 h in the presence or absence of SB203580 (1 µM) or SP600125 (5 µM) and the effect on A2AR mRNA expression was detected. The results are expressed as percentage of IL-1β- or TNF-{alpha}-stimulated A2AR mRNA. *, p < 0.05; **, p < 0.01vs IL-1β- or TNF-{alpha}-stimulated cells; #, p < 0.05; ##, p < 0.01 vs control unstimulated cells, n = 6 except for kinase assays where n = 3.

 
Effect of IL-1β and TNF-{alpha} on A2AR mRNA degradation

Control of gene expression by proinflammatory cytokines like IL-1β and TNF-{alpha}, as well as different forms of stress may be regulated, at least in part, by an increase in half-life of the transcripts (46, 47, 48). To reveal whether the induction resulted from posttranscriptional stabilization of A2AR mRNA, we conducted transcriptional blockade studies. The RNA polymerase II inhibitor actinomycin D (5 µg/ml) was added to A549 cell culture 2 h after IL-1β (10 ng/ml) or TNF-{alpha} (10 ng/ml) stimulation. The A2AR mRNA levels were analyzed by quantitative real-time PCR after 0, 0.5, 1, 2, and 4 h of actinomycin D treatment, and the amount of A2AR mRNA relative to GAPDH mRNA degraded per hour was measured. In studies where no IL-1β or TNF-{alpha} was added before actinomycin D treatment, the amount of A2AR mRNA was very low whereas stimulation for 2 h induced an approximate 4-fold induction of A2AR mRNA by both stimuli. We were unable to show any effect of IL-1β or TNF-{alpha} on the A2AR mRNA degradation rate (Fig. 11). Addition of actinomycin D before cell stimulation also prevented induction of A2AR mRNA induced by either IL-1β or TNF-{alpha} without affecting the rate of mRNA degradation (data not shown), suggesting that both treatments do affect de novo transcription.


Figure 11
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  		FIGURE 11. Rate of A2AR mRNA degradation. Cells were pretreated with IL-1β (10 ng/ml) or TNF-{alpha} (10 ng/ml) or vehicle for 2 h (–2 h to 0). Actinomycin D (5 µg/ml) was added at time 0 and A2AR mRNA levels were determined after 0, 0.5, 1, 2, and 4 h by quantitative real-time RT-PCR and expressed as a ratio to that of GAPDH. The rate of mRNA degraded is expressed as the change in A2AR:GAPDH ratio per hour.

 
Effect of A2AR stimulation on cytokine and anti-inflammatory mediator release

To investigate whether induction of A2AR expression by IL-1β and TNF-{alpha} resulted in a functional effect of receptor stimulation, A549 cells were stimulated with IL-1β (1 ng/ml) or TNF-{alpha} (10 ng/ml) for 24 h. Cells were then restimulated with IL-1β or TNF-{alpha} in the presence of the A2AR agonist CGS21680 and the expression of the proinflammatory gene GM-CSF and the anti-inflammatory gene SLPI (secretory leukocyte protease inhibitor) was measured. CGS21680 attenuated both IL-1β- and TNF-{alpha} -induced GM-CSF release (Fig. 12, a and b), whereas the expression of SLPI was enhanced (Fig. 12, c and d). In contrast, CGS21680 had no effect on previously unstimulated cells (data not shown). These data suggest that proinflammatory cytokines can induce the expression of functional A2AR.


Figure 12
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  		FIGURE 12. Functional effect of A2AR agonist on GM-CSF and SLPI gene expression. A549 cells were stimulated with IL-1β (1 ng/ml) or TNF-{alpha} (10 ng/ml) for 24 h to induce A2AR expression before restimulation of cells with IL-1β or TNF-{alpha} for 6 h. The effect of the A2AR agonist CGS21680 (1 µM) on GM-CSF and SLPI release is shown. The results are the mean of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we report that the expression of the A2A receptor in human epithelial A549 cells is up-regulated within 24 h at the mRNA and protein level by IL-1β and TNF-{alpha}, but not by LPS, by distinct mechanisms. A similar degree of A2AR mRNA induction was seen in primary human airway epithelial cells. Both TNF-{alpha} and IL-1β required IKK2, p38 MAPK, and JNK activation to induce A2AR expression. However, whereas IL-1β-stimulated A2AR mRNA expression was dependent upon activation and binding of p65 to {kappa}B-REs within the native proximal A2AR promoter, despite clear activation of NF-{kappa}B by TNF-{alpha} in these cells confirming previous results (49), native {kappa}B-REs are not readily occupied following TNF-{alpha} stimulation, at least within 4 h, by which time A2AR mRNA expression is already substantially enhanced. This differential effect was confirmed using an A2AR promoter-luciferase reporter gene and importantly we were able to show that neither IL-1β nor TNF-{alpha} had any effect on the rate of A2AR mRNA degradation.

NF-{kappa}B is an important regulator of the inflammatory and immune responses in mammalians cells (50, 51). NF-{kappa}B is activated in a variety of cell types, and its activation leads to the rapid induction of a wide range of genes involved in inflammatory and acute responses, in the function and development of the immune system, apoptosis, cell-cycle regulation, and development (50, 52). Consistent with the notion of NF-{kappa}B involvement in IL-1β- and TNF-{alpha}-induced expression of A2AR mRNA, the IKK2 inhibitor AS602868 attenuated IL-1β- and TNF-{alpha}-induced A2AR mRNA expression with a similar IC50. AS602868 is a selective inhibitor of IKK2 with an IC50 of ~1 µM in cell-based assays (53); however, at high concentrations, the compound also shows some inhibitory effect on JNK2 and p38 MAPK (53). Therefore, we examined whether the differential effect of IL-1β and TNF-{alpha} on the induction of A2AR expression was a result of MAPK activation.

It is clear that in mammalian cells, external stressors and cytokines act through signaling pathways other than the NF-{kappa}B pathway (52), including the ERK, JNK, and p38 MAPK pathways (54, 55). The ERK cascade is characteristically activated by various growth factors and is critical for proliferation and survival (56), whereas the JNK and p38 MAPK pathways are stimulated by the proinflammatory cytokines TNF-{alpha} and IL-1β, as well as stress stimuli, such as heat shock UV radiation and hyperosmolarity (57, 58). The p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125 were also able to reduce A2AR mRNA levels induced by both cytokines. The specificity of the JNK inhibitor SP600125 has recently been questioned (59) and at high concentrations (>25 µM) it can inhibit p38 and other related MAPKs (60). However, since we observed significant inhibition of A2AR expression at 5 µM, our results can probably be attributed to a selective inhibition of JNK. Furthermore, since inhibition of either the p38 MAPK and JNK pathways attenuated A2AR expression to a similar extent after each stimulus, the data suggest that the differential effects of IL-1β and TNF-{alpha} are not a result of MAPK activation.

Despite the potential limitations of transient reporter gene assays, we were able to confirm a functional role for some of the sites occupied by p65 using the ChIP assay. We demonstrated that the {kappa}B site II was essential for NF-{kappa}B regulation of A2AR promoter activity stimulated by IL-1β. These data also confirmed the ChIP data with respect to the actions of TNF-{alpha} in that TNF-{alpha} was not able to stimulate the A2AR promoter. This lack of effect of TNF-{alpha} on the A2AR reporter gene assay also suggested that despite RelB being activated by TNF-{alpha} with the required time course, it was unlikely to be playing a role in TNF-{alpha}-mediated transcriptional induction of A2AR. It leaves the possibility that the {kappa}B binding sites utilized by TNF-{alpha} reside outside of the classic proximal promoter/enhancer region as seen, for example, with the human NO synthase 2 (61) and SLPI genes (62).

Our study also demonstrates that A2AR mRNA is not regulated at the level of mRNA degradation following cytokine stimulation. Many studies examining mRNA stability ignore the large differences in starting levels of mRNA and indeed in this study merely examining the time to show 50% degradation of mRNA indicated a difference in half-life between IL-1β- and TNF-{alpha}-stimulated cells. However, taking into account the differences in starting mRNA levels and determining the rate of degradation of mRNA, no differences could be seen.

Previous reports in other cell types have shown that A2AR mRNA and/or protein expression can be induced by TNF-{alpha} and IL-1β. Using semiquantitative RT-PCR and Western blotting, Khoa et al. (31) showed that TNF-{alpha}- and IL-1β-induced A2AR mRNA was stimulated by 50% at 3 h in human monocytic THP-1 cells, and that this small induction was maintained for up to 18 h. This increase in mRNA expression was accompanied by a 30% increase in A2AR protein expression after overnight incubation (31). The same group reported a similar time course and degree of A2AR mRNA induction by TNF-{alpha} and IL-1β in human microvascular endothelial cells (63). Interestingly, in these experiments, a 2-fold greater induction of A2AR mRNA was reported when quantitative RT-PCR was used. This again was accompanied by a 30% increase in A2AR protein expression seen after overnight stimulation. Furthermore, both IL-1β and TNF-{alpha} were shown to induce A2AR receptor mRNA and protein expression in rat PC12 cells (32) using semiquantitative RT-PCR and ligand-binding assays. A small (35–50%) induction of mRNA was reported with TNF-{alpha} which was sustained from 3 to 48 h and correlated with a 50% increase in protein at 3 h, although this increased over the 48 h of the experiments. Similar results were reported for IL-1β-induced A2AR expression.

In contrast, Fortin et al. (64) reported a 4- to 5-fold increase in A2AR mRNA expression after 4–6 h of IL-1β and TNF-{alpha} treatment of human neutrophils. The induction of mRNA was associated with a 2- to 2.5-fold increase in protein expression at 4 and 12 h after TNF-{alpha} but with no induction following IL-1β stimulation at any time point investigated (64). In addition, TNF-{alpha} induced a 2- to 3-fold increase in A2AR mRNA and protein expression in PBMCs as measured by semiquantitative RT-PCR from normal subjects but not those with congestive heart failure (65). This may reflect the fact the enhanced basal expression of A2AR mRNA and protein expression reported in PBMCs from patients with congestive heart failure (65). We have also reported increased expression of A2AR mRNA and protein in lung tissue from patients with COPD (36). Overall, the data in several cell types suggest that both TNF-{alpha} and IL-1β can induce A2AR mRNA and protein expression, although the time course and degree of induction varies depending upon cell type and, for mRNA determination, the method of detection used. Evidence also suggests that control of A2AR protein expression must be differentially regulated in these cells since in some cells receptor expression is increased concomitant with mRNA and in others it is delayed and increases between 24 and 48 h (32), and this may occur here in epithelial cells with greater protein induction resulting at time points greater than 24 h.

In conclusion, these results suggest that distinct actions of NF-{kappa}B are involved in the regulation of A2AR gene expression by IL-1β and TNF-{alpha}. Thus, although NF-{kappa}B is essential for the regulation of A2AR gene transcription by IL-1β and TNF-{alpha}, the precise sites within the A2AR promoter/enhancer region utilized are distinct. The actions of NF-{kappa}B activated by TNF-{alpha} may require binding to an alternative {kappa}B-RE outside the classic proximal promoter targeted by IL-1β-activated NF-{kappa}B. Although the biological significance of this increased expression on epithelial cells is not yet known, our results support the hypothesis that the A2AR might play a role in the pathogenesis of inflammatory bronchopulmonary disease.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by GlaxoSmithKline (U.K.). Back

2 Address correspondence and reprint requests to Dr. Ian M. Adcock, Airways Disease Section, Guy Scadding Building, National Heart & Lung Institute, Imperial College London, Dovehouse Street, London, SW3 6LY, U.K. E-mail address: ian.adcock{at}imperial.ac.uk Back

3 Abbreviations used in this paper: COPD, chronic obstructive pulmonary disease; IKK2, I{kappa}B kinase; SLPI, secretory leukocyte protease inhibitor; ChIP, chromatin immunoprecipitation. Back

Received for publication January 31, 2006. Accepted for publication August 31, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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