Introduction

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The endogenous nucleoside adenosine can be released to or formed in the extracellular space under hypoxic and inflammatory conditions. Once generated, adenosine acts as an autocoid by interacting with adenosine receptors belonging to the seven transmembrane G protein-coupled group of cell surface receptors. Among other sources, activated mast cells, platelets, and neutrophils have been shown to release adenosine and adenine nucleotides. Adenosine has diverse effects on inflammatory process. The activation of A_2A receptors inhibits oxidative burst, degranulation, and adhesion of neutrophils (Cronstein et al., 1992) and also inhibits platelet aggregation (Cristalli et al., 1994). Adenosine at A₁ receptors can increase neutrophil chemotaxis (Cronstein et al., 1992), whereas activation of A₃ receptors inhibits eosinophil chemotaxis (Walker et al., 1997).

The ability of extracellular adenosine to modulate mast cell function has long been recognized (Marquardt et al., 1978; Church and Hughes, 1985; Peachell et al., 1988). Activation of mast cells by adenosine has been implicated in the pathophysiology of asthma (Church and Holgate, 1986; Feoktistov et al., 1998). Inhaled adenosine, or its precusor AMP, provokes bronchoconstriction in asthmatic patients via activation of mast cells (Cushley and Holgate, 1985). Whereas adenosine A₃ receptor has been shown to modulate rat mast cell function (Ramkumar et al., 1993), it appears that the A_2B adenosine receptor subtype regulates mouse (Marquardt et al., 1994), canine (Auchampach et al., 1997), and human mast cell activation (Feoktistov and Biaggioni, 1995).

We previously showed that the human mast cell line HMC-1 expresses functional A_2A and A_2B receptors (Feoktistov and Biaggioni, 1995; Feoktistov and Biaggioni, 1998). Both A₂ subtypes of adenosine receptors activate adenylate cyclase via G_s-protein. However, only the A_2B receptor has been shown to also be coupled to phospholipase C in mast cells via a GTP-binding protein of the G_q family. Furthermore, the nonselective A_2A/A_2B adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), but not the selective A_2A agonist 4-((N-ethyl-5'-carbamoyladenos-2-yl)-aminoethyl)-phenylpropionic acid (CGS 21680), induced secretion of interleukin-8 (IL-8) from HMC-1 cells (Feoktistov and Biaggioni, 1995). IL-8 is not stored preformed in HMC-1 and requires synthesis de novo (Selvan et al., 1994), suggesting that stimulation of adenosine A_2B receptors can trigger transcription and synthesis of this cytokine. However, the mechanism by which adenosine promotes biosynthesis of IL-8 is not clear.

Recently, several mitogen-activated protein kinase (MAPK) signaling pathways have been demonstrated to play a central role in mediating intracellular signal transduction from the cell surface to the nucleus. At least three MAPK subfamilies are present in mammalian cells and form distinct signaling cascades. These include extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 MAPKs. There is recent evidence that the p38 MAPK pathway regulates synthesis of IL-8 in neutrophils stimulated with tumor necrosis factor- and N-formylmethionine leucyl-phenylalanine, whereas the ERK cascade does not play any role in this process (Zu et al., 1998). Less is known about the coupling of adenosine receptors to MAPK pathways. It has been shown that adenosine A_2A receptors expressed in Chinese hamster ovary cells inhibit thrombin-induced ERK activation via increases in cAMP (Hirano et al., 1996). Similarly, the selective A_2A agonist CGS 21680 inhibited ERK activation by cAMP-dependent mechanism in mast cells derived from human umbilical cord blood (Suzuki et al., 1998). In contrast, in human endothelial cells A_2A adenosine receptors stimulated ERK pathway independently from cAMP (Sexl et al., 1997). The coupling of A_2B adenosine receptors with MAPK signaling pathways, to our knowledge, has not been described and the coupling of A₂ adenosine receptors to JNK and p38 MAPKs has not been reported. The present study was undertaken to elucidate the role of MAPK signaling pathways in A_2B-mediated IL-8 secretion in the human mast cell line HMC-1. We report that stimulation of adenosine A₂ receptors triggers activation of p21^ras-ERK, JNK, and p38 MAPK signaling pathways. Using highly selective inhibitors of extracellular signal-regulated protein kinase kinase (MEK) and p38 MAPK we found that stimulation of ERK and p38 MAPK pathways are essential steps in the mechanism of adenosine A_2B receptor-mediated IL-8 secretion in HMCs.

	Materials and Methods

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Cell Culture and Reagents. HMC-1 cells were a generous gift from Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN). HMC-1 cells were maintained in suspension culture at a density between 3 and 9 × 10⁵ cells/ml by dilution with Iscove's medium supplemented with 10% (vol/vol) fetal bovine serum, 2 mM glutamine, antibiotics, and 1.2 mM -thioglycerol. The cells were kept under a humidified atmosphere of air/CO₂ (19:1) at 37°C.

Measurement of IL-8 Secretion. HMC-1 cells were harvested and resuspended to a concentration of 10⁶ cells/ml in serum-free Iscove's media containing 1 U/ml adenosine deaminase. Cells were incubated for 2.5 h (or for the times indicated in Results) under a humidified atmosphere of air/CO₂ (19:1) at 37°C with the reagents indicated in Results. At the end of this incubation period, the culture media were collected by centrifugation at 12,000g for 1 min at 4°C. IL-8 concentrations were measured using an enzyme-linked immunosorbent assay kit (American Laboratory Products Co. Ltd, Windham, NH).

Stimulation of Mast Cells. HMC-1 cells were harvested and resuspended to a concentration of 10⁷ cells/ml in a buffer, pH 7.4, containing 150 mM NaCl, 2.7 mM KCl, 0.37 mM NaH₂PO₄, 1 mM MgSO₄, 1 mM CaCl₂, 5 g/liter D-glucose, 10 mM HEPES-NaOH, and 1 U/ml adenosine deaminase. After 15 min preincubation at 37°C, 200- to 1000-µl aliquots of cell suspension were incubated for various times at 37°C with the reagents indicated in Results.

Assay of Tyrosine Phosphorylation of ERK. To evaluate phosphorylation of ERK at tyrosine residues, following each stimulation, 2 × 10⁶ HMC-1 cells were collected by centrifugation for 15 s at 12,000g and lysed by addition of 100 µl of boiling 1% SDS. After boiling for 15 min, samples were diluted with 900 µl of ice-cold buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 2 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride (lysis buffer A). Unsoluble material was removed by centrifugation for 10 min at 16,000g at 4°C and supernatant was precleared with 50 µl of agarose for 1 h at 4°C. Proteins phosporylated on tyrosine residues were precipitated after incubation for 4 h at 4°C with antiposphotyrosine monoclonal antibody (clone PT-66) covalently coupled to agarose (Sigma Chemical Co.). The immune complexes were washed three times with the lysis buffer A, and then 50 µl of sample buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 10% glycerol, 5% -mercaptoethanol, 0.5% bromophenol blue) was added. After boiling for 5 to 10 min, the samples (20 µl) were loaded onto 0.75-mm 10% SDS-polyacrylaminde gel electrophoresis (PAGE) gels, and discontinuous electrophoresis was performed as described by Laemmli (1970). Proteins on the gel were transferred to Immobilon-P polyvinilidene fluoride 0.45-µm membrane (Millipore, Bedford, MA). Nonspecific protein binding sites on the membrane were blocked by incubation for 2 h at room temperature or overnight at 4°C in 5% (w/v) skimmed milk powder, 0.2% (v/v) Tween-20, 100 mM Tris-HCl, pH 7.5, and 0.9% (w/v) NaCl. ERK1/ERK2-specific antiserum (M5670, Sigma Chemical Co.) was incubated with the membrane for 1 h at room temperature at a dilution of 1:1000 in a fresh blocking solution. The blot was then washed five times with 0.2% (v/v) Tween-20, 100 mM Tris-HCl, pH 7.5, and 0.9% (w/v) NaCl (10 min/wash) and then incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (Sigma Chemical Co.) in the blocking solution. The membrane was washed again as described above and the bands were visualized with an enhanced chemiluminescence method (Nesbitt and Horton, 1992).

Assay of ERK Enzymatic Activity. To measure activity of ERK, following each stimulation, 2 × 10⁶ HMC-1 cells were collected by centrifugation for 15 s at 12,000g and lysed by adding 70 µl of the lysis buffer A, containing also 5 µg/ml pepstatin and 2 µM microcystin. After incubation in an ice bath for 1.5 h, insoluble material was removed from lysates by centrifugation at 16,000g for 10 min at 4°C. Affinity-purified polyclonal ERK1/ERK2-specific antibody R2 Erk 1-CT (Upstate Biotechnology, Lake Placid, NY) was preincubated with protein A-agarose (Pharmacia Biotech Inc., Piscataway, NJ) at a ratio of 1 µg IgG per 30 µl agarose in the lysis buffer A for 1 h at 4°C, then washed three times with the same buffer and combined with 50 µl of lysates. Enzymatically active ERK was precipitated after incubation for 2 h at 4°C. The immune complexes were washed twice with the lysis buffer A and once with assay dilution buffer, containing 20 mM 3-(N-morpholino)propanesulfonic acid, pH 7.2, 25 mM -glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, and 1 mM dithiothreitol. ERK activity was evaluated with a MAPK assay kit (Upstate Biotechnology) according to the manufacturer's protocol.

Evaluation of JNK and p38 MAPK Activation. Dual phosphorylation of MAPKs at both threonine and tyrosine residues in a regulatory Thr-Xaa-Tyr site has been shown to be an accurate indicator of their activation (Burack and Sturgill, 1997; Moriguchi et al., 1996; Raingeaud et al., 1995). To evaluate the dual phosphorylation of JNK and p38 MAPKs, following each stimulation, 2 × 10⁶ HMC-1 cells were collected by centrifugation for 15 s at 12,000g and lysed by addition of 100 µl sample buffer (250 mM Tris-HCl, pH 6.8; 10% SDS, 10% glycerol, 5% -mercaptoethanol, and 0.5% bromophenol blue). After 2 s of sonication, samples were heated to 95 to 100°C for 5 min and insoluble material was removed by centrifugation at 16,000g for 10 min at 4°C. The samples (20 µl) were loaded onto 0.75-mm 10% SDS-PAGE gels, and discontinuous electrophoresis was performed as described by Laemmli (1970). Western blotting was done as described above. Affinity-purified antibody specific for human p38 MAPK phosphorylated at Thr¹⁸⁰ and Tyr¹⁸² (Calbiochem-Novabiochem Corp.) or monoclonal IgG₁ antibody specific for human JNK phosphorylated at Thr¹⁸³ and Tyr¹⁸⁵ (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was incubated with the membrane for 1 h at room temperature in a fresh blocking solution. The blot was then washed five times with 0.2% (v/v) Tween-20, 100 mM Tris-HCl, pH 7.5, 0.9% (w/v) NaCl (10 min/wash) and then incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (Sigma Chemical Co.) or with horseradish peroxidase-conjugated anti-mouse IgG (Sigma Chemical Co.) in the blocking solution. The membrane was washed again as described above and the bands were visualized with an enhanced chemiluminescence method (Nesbitt and Horton, 1992).

Evaluation of p21^ras Activation. The active GTP-bound form of p21^ras was detected using the minimal p21^ras-binding domain of Raf-1 (RBD) according to previously published technique (de Rooij and Bos, 1997). pGEX 2T RBD prokaryotic expression vector was kindly provided by Dr. A. Wittinghoffer (Max-Planck Institute, Dortmund, Germany). Glutathione S-transferase (GST)-RBD was produced and isolated as described (Herrmann et al., 1995). Following each stimulation, 10⁷ HMC-1 cells were collected by centrifugation for 15 s at 12,000g and lysed by addition of 200 µl 6 mM Na₂HPO₄, 4 mM NaH₂PO₄, 1% NP-40, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.1 mM Na₃VO₄, 4 µg/ml leupeptin, and 2 mM benzamidine, containing Complete Mini protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany) (lysis buffer B). GST-RBD, precoupled to glutathion-agarose beads in the lysis buffer B, was added and lysates were incubated at 4°C for 30 min. Beads then were washed five times with the lysis buffer B by centrifugation and resuspended in 40 µl of sample buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 10% glycerol, 5% -mercaptoethanol, and 0.5% bromophenol blue). After boiling for 5 to 10 min, the supernatant was collected by centrifugation and the protein samples (20 µl) were separated on a 15% SDS-PAGE gel and subsequently transferred to polyvinilidene fluoride membrane by Western blotting. p21^ras was detected by incubating the membrane with the rat monoclonal antibody Y13-259 (Calbiochem-Novabiochem Corp.) overnight at 4°C. Horseradish peroxidase-conjugated anti-rat IgG (Sigma Chemical Co.) was used as a second antibody (1 h at room temperature). The bands on the membrane were visualized with an enhanced chemiluminescence method (Nesbitt and Horton, 1992).

Measurement of Intracellular Calcium. Cytosolic free calcium concentrations were determined by fluorescent dye techniques. HMC-1 cells (2 × 10⁶ cells/ml) were loaded with 1 µM fura-2/acetoxymethyl ester in a buffer containing 150 mM NaCl, 2.7 mM KCl, 0.37 mM NaH₂P0₄, 1 mM MgSO₄, 1 mM CaCl₂, 5 g/liter D-glucose, 10 mM HEPES-NaOH, pH 7.4, and 0.35% BSA. After incubation for 1 h at room temperature, cells were washed to remove excess of fura-2 and were resuspended (2 × 10⁵ cells/ml) in the same buffer containing 1 U/mL adenosine deaminase and no BSA. Fluorescence was monitored at an emission wavelength of 510 nm and excitation wavelengths of 340 and 380 nm. Maximal fluorescence was determined after addition of 0.004% digitonin. Minimal fluorescence was then determined in the presence of 20 mM EGTA. The intracellular calcium was calculated using previously described formulae (Grynkiewicz et al., 1985), assuming a K_d of 224 nM. Fluorescence was measured with a spectrofluorimeter (Fluorolog 2; Spex Industries, Inc., Edison, NJ) in a thermostated cuvette (37°C).

	Results

Top Summary Introduction Materials and Methods Results Discussion References

Adenosine Induces Tyrosine Phosphorylation and Activation of ERK. The activation of ERK1/ERK2 is accompanied by the phosphorylation of Tyr¹⁸⁵ residue at the TEY motif (Burack and Sturgill, 1997), which can be detected using immunoprecipitation with antiphosphotyrosine antibody following by immunoblotting with specific anti-ERK1/ERK2 antibody. To determine whether adenosine induces tyrosine phosphorylation of ERK isoforms, HMC-1 cells were incubated for 5 or 30 min with the stable adenosine analog NECA at a concentration of 100 µM. Cells were incubated in the presence of 1 U/ml of adenosine deaminase to remove endogenously produced adenosine. PMA, a potent activator of protein kinase C, was used as a positive control at the concentration of 10 nM. As seen in Fig. 1, NECA, and to a greater extent PMA, induced phosphorylation of both the p42 and p44 isoforms of ERK. This effect was evident after 5 min of incubation. After 30 min of incubation in the presence of NECA, the tyrosine phosphorylation of ERK was completely reversed, and it was also considerably reduced in the presence of PMA.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Tyrosine phosphorylation of ERK isoforms in HMC-1. Cells were incubated in the absence or presence of 100 µM NECA for 5 and 30 min; 10 nM PMA was used as a positive control. Proteins phosphorylated on tyrosine residues were immunoprecipitated from cell lysates with a phosphotyrosine antibody. After separation on 10% SDS-PAGE, phosphorylated ERK isoforms were identified by immunoblotting with antiserum against p42/p44 ERK.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Stimulation of ERK activity in HMC-1. Cells were incubated for 1 min in the absence (CON) or presence of 10 µM NECA (NECA), 10 µM CGS 21680 (CGS), 100 µM forskolin (FORSK), or 10 nM PMA (PMA). ERK was immunoprecipitated from cell lysates with an antibody against p42/p44 ERK, and enzymatic activity was determined by phosphorylation of myelin basic protein. Normalized values are presented as mean ± SEM of four independent experiments done in triplicate.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Effect of 10 µM NECA, 10 nM calcium ionophore A23187, and 10 nM thapsigargin on free intracellular calcium and ERK activation in HMC-1. Upper panel, rise in inracellular calcium was determined by fura-2 technique within 1 min after addition of vehicle (V), NECA (N), A23187 (A), or thapsigargin (T). Results are presented as mean ± SEM of four experiments. Lower panel, activation of ERK was evaluated by immunoblotting with phospho-specific ERK antibody within 1 min after stimulation. These results are representative of three similar experiments.

Stimulation of p21^ras by Adenosine. Increase in guanine nucleotide exchange on p21^ras results in binding of this small G protein to Raf protein kinase with subsequent stimulation of MEK and ERK activation. To determine whether adenosine induces formation of GTP-bound active form of p21^ras, we incubated cells with the stable adenosine analog NECA (10 µM) in the presence of 1 U/ml adenosine deaminase. Samples were collected at different time points and the extracted proteins were incubated with bacterially expressed GST-p21^ras-binding domain of Raf coupled to glutathion-agarose. The absorbed proteins were then analyzed by immunoblotting with anti-p21^ras antibody. As seen in Fig. 4, the nonselective A_2A/A_2B agonist NECA induced maximal formation of active p21^ras during the first minute, whereas the selective A_2A agonist CGS 21680 produced virtually no effect.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Time course of activation of p21ras produced by 10 nM PMA, 10 µM NECA, and 1 µM CGS 21680 (CGS) in HMC-1 cells. Effects of PMA and NECA were determined at indicated periods of time, and effect of CGS 21680 was determined at 1 min only. Analysis of p21ras activation was performed by Western blotting of proteins absorbed to GST-p21ras-binding domain of Raf as described in Materials and Methods. These results are representative of three similar experiments.

Adenosine Activates JNK and p38 MAPK. To determine whether adenosine stimulation of HMC-1 cells would trigger activation of other MAPK cascades, we incubated cells with the stable adenosine analog NECA (10 µM) in the presence of 1 U/ml adenosine deaminase. Samples were collected at different time points and immunoblotted either with an antibody specific for human p38 MAPK phosphorylated at Thr¹⁸⁰ and Tyr¹⁸² or with an antibody specific for human JNK phosphorylated at Thr¹⁸³ and Tyr¹⁸⁵. The double phosphorylation of the TGY motif in p38 MAPK or TPY in JNK is considered to be an accurate indicator of their stimulation (Raingeaud et al., 1995; Moriguchi et al., 1996). As seen in Fig. 5, the stable adenosine analog NECA stimulated both JNK and p38 MAPK. It should be noted, however, that stimulation of these kinases followed different kinetics; maximal stimulation of p38 MAPK was observed within the first minute, whereas JNK activation reached a maximum only within 10 to 15 min of incubation with NECA. The JNK antibody used in these experiments recognizes the phosphorylated p55 and p46 isoforms of JNK, but with NECA we consistently observed an increase in the band corresponding to p46 JNK only. In some but not all gels we observed an appearance of a faint band of a lower molecular weight, which may be explained by phosphorylation of a product of partial proteolytic degradation of JNK. The data, presented here, confirm that adenosine, in parallel with the activation of ERK cascade shown above, can also stimulate JNK and p38 MAPK pathways in HMC-1 cells.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Time course of JNK and p38 MAPK activation by NECA in HMC-1. Cells were incubated with 10 µM NECA for indicated periods of time. Analysis of JNK and p38 MAPK activation was performed concomitantly in the same samples. Proteins from cell lysates were separated on 10% SDS-PAGE. MAPKs activation was determined by immunoblotting with antibody specific for phosphorylated JNK or for phosphorylated p38 MAPK. These results are representative of four similar experiments.

Stimulation of ERK and p38 MAPK Cascades Is Required for Adenosine A_2B Receptor-Mediated IL-8 Secretion. We previously showed that stimulation of adenosine A_2B receptors in HMC-1 cells induces secretion of IL-8 (Feoktistov and Biaggioni, 1995). Figure 6A shows that there is a 1-h delay between stimulation with 100 µM NECA and secretion of IL-8, the time probably needed to initiate protein synthesis. IL-8 production increased linearly between 1 and 3 h of incubation with NECA and reached maximal levels of 33 ± 0.5 pmol/ml after 4 h. In a control experiment spontaneous release of IL-8 reached 4 ± 0.2 pmol/ml after 4 h of incubation in the absence of NECA (Fig 6A). Incubation of HMC-1 cells with increasing concentrations of NECA for 2.5 h revealed a sigmoid curve for IL-8 production with an EC₅₀ of 3 µM (Fig. 6B). In parallel experiments we also made an approximate estimate of the effects of increasing concentrations of NECA on activation of ERK and p38 MAPK. As seen in the inset of Fig. 6B, NECA in micromolar concentrations produced a dose-dependent phosphorylation of the TXY motif, measured as an increase in the intensity of the immunoreactive p44 ERK and p38 MAPK bands in corresponding immunoblots. The relatively low potency of NECA agrees with previous reports of A_2B receptor-mediated IL-8 production in HMC-1 cells (Feoktistov and Biaggioni, 1995).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Release of IL-8 from HMC-1 cells. A, time course of IL-8 release from the cells stimulated with 100 µM NECA (). , Spontaneous release of IL-8 after 4 h of incubation in the absence of NECA. B, effect of increasing concentrations of NECA on IL-8 release from the cells incubated for 2.5 h. Values are presented as mean ± SEM of four experiments. Inset, effect of increasing concentrations of NECA on activation of p38 MAPK and p44 ERK. MAPK activation was evaluated by increase in intensity of phospho-specific immnoreactive bands in corresponding Western blots, using SigmaScan/Image software (Jandel Scientific, San Rafael, CA). Results represent an average of two experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Effect of selective protein kinase C inhibitor Ro 32-0432 on IL-8 release stimulated by 3 µM NECA or by 3 nM PMA for 2.5 h. Values are presented as mean ± SEM of three experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Effect of tyrosine kinase inhibitors genistein (A) and herbimycin A (B), phosphatidylinositol 3-kinase inhibitor wortmannin (C), selective MEK inhibitor PD 098058 (D), and selective p38 MAPK inhibitors SB 202190 (E) and SB 203580 (F) on A2B adenosine receptor-dependent IL-8 release from HMC-1 cells. HMC-1 cells were incubated with 3 µM NECA for 2.5 h in the absence or presence of increasing concentrations of these inhibitors. Values are presented as mean ± SEM of three or four experiments.

	Discussion

Top Summary Introduction Materials and Methods Results Discussion References

Four distinct subtypes of adenosine receptors, specifically A₁, A_2A, A_2B, and A₃, have been pharmacologically characterized and cloned. The classification of adenosine receptors was originally based on their ability to modulate intracellular levels of cAMP. A₂ adenosine receptors stimulate adenylate cyclase through coupling with G_s protein, whereas A₁ and A₃ adenosine receptors are coupled to adenylate cyclase via inhibitory GTP-binding proteins of the G_i/o family. Although activation of adenylate cyclase is arguably an important signaling mechanism for A_2A receptors, this is not necessarily the case for A_2B receptors, because other intracellular pathways have been found to be functionally coupled to A_2B receptors in addition to adenylate cyclase. We previously showed that the human mast cell line HMC-1 expresses functional A_2A and A_2B receptors, whereas no evidence was found for A₁ or A₃ adenosine receptors (Feoktistov and Biaggioni, 1995, 1998). Both A₂ subtypes of adenosine receptors activate adenylate cyclase via G_s protein. However, only A_2B receptor was found to couple also to phospholipase C via a GTP-binding protein of the G_q family (Fig. 9). A similar coupling of adenosine A_2B receptors to phospholipase C has been also found in mouse bone marrow-derived mast cells (Marquardt et al., 1994) and in the canine BR mast cell line (Auchampach et al., 1997).

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Schematic representation of intracellular pathways coupled to adenosine receptors in HMC-1. We previously demonstrated that HMC-1 cells express functional A2A (A2AAR) and A2B (A2BAR) receptors. Both subtypes activate adenylate cyclase (AC) via Gs protein. Activation of this pathway results in accumulation of cAMP and stimulation of protein kinase A (PKA). However, only A2B receptors are coupled also to phospholipase C (PLC) via a GTP binding protein of the Gq family. Activation of this pathway results in an increase in diacylglycerol (DAG) and IP3. Diacylglicerol stimulates PKC. Inositol trisphosphate activates mobilization of calcium from intracellular stores. Stimulation of A2B receptors eventually leads to production of IL-8 (Feoktistov and Biaggioni, 1995). In this study we present evidence that stimulation of A2B receptors activates the small GTP-binding protein p21ras. This event triggers ERK signaling pathway with sequential stimulation of Raf, MEK1/2, and ERK1/2 protein kinase activities. The exact mechanism of coupling of A2B receptors with ERK pathway remains to be determined. This may include combination of activation of p21ras via Gq (Watanabe et al., 1995; Eguchi et al., 1996) and stimulation of Raf via PKC (Hawes et al., 1995) (indicated by a question mark). We also demonstrate the coupling of adenosine receptors to JNK and p38 MAPK signaling pathways, but the upstream events have not been explored. Blockade of ERK pathway with the selective MEK inhibitor PD 098058 or p38 MAPK pathway with the selective p38 MAPK inhibitors SB 202190 and SB 203580 revealed that these MAPK pathways are essential for adenosine A2B receptor-mediated production of IL-8.

We previously demonstrated that stimulation of adenosine receptors with the nonselective A_2A/A_2B agonist NECA but not the selective A_2A agonist CGS 21680 induced production of IL-8 from HMC-1 cells (Feoktistov and Biaggioni, 1995). These data indicate that A_2B receptors stimulate intracellular pathways eventually leading to transcription and synthesis of IL-8. We hypothesized that among possible candidates for these pathways were MAPK cascades serving as information relays connecting cell-surface receptors to nuclear transcription factors. In favor of this hypothesis it was reported recently that p38 MAPK but not ERK pathway is involved in tumor necrosis factor- and N-formylmethionine leucyl-phenylalanine-stimulated induction of IL-8 in neutrophils (Zu et al., 1998).

The novel finding reported here is that A_2B receptors are coupled to the ERK pathway (Fig. 9). This is based on the observation that the nonselective A_2A/A_2B agonist NECA but not the selective A_2A agonist CGS 21680 stimulates p21^ras and activates ERK. The current pharmacological characterization of A_2B receptors relies on the lack of effectiveness of compounds that are potent and selective agonists of other receptor types (Feoktistov and Biaggioni, 1997). In HMC-1 cells, where only A_2A and A_2B receptors are present (Feoktistov and Biaggioni, 1995), the lack of effect of the selective A_2A agonist CGS 21680 on ERK activity indicates an A_2B-mediated mechanism. We also examined whether the second messengers, known to be generated upon activation of A_2B receptors, would be responsible for activation of ERK signaling pathway by NECA. Stimulation of both A_2B and A_2A receptors in HMC-1 cells results in increase of cAMP. However, the increase of cAMP produced by forskolin failed to mimic the effect of NECA on ERK activity. In addition to stimulation of adenylate cyclase in HMC-1 cells, A_2B receptors also activate phospholipase C, resulting in mobilization of intracellular calcium and generation of diacylglycerol, a natural activator of protein kinase C (Feoktistov and Biaggioni, 1995). It has been previously reported that thapsigargin and A23187 can stimulate ERK activity in epidermal cells and fibroblasts, but this effect was not observed within the first minute and was obvious only after a 4- to 5-min delay (Chao et al., 1992). Because stimulation of ERK by NECA reached its maximum within 1 min, we focused on the early effects of thapsigargin and A23187 on intracellular calcium and ERK activation in HMC-1 cells in our study. Stimulation of calcium mobilization with thapsigargin or generation of calcium influx with calcium ionophore A23187 followed closely the kinetics of the NECA-dependent calcium increase within the first minute, but they failed to mimic the activation of ERK by NECA. In contrast, stimulation of protein kinase C with PMA produced a strong activation of ERK within the first minute, comparable with the effects of NECA. The mechanism of stimulation of ERK pathway by protein kinase C remains unclear, and may include a p21^ras-dependent (Marais et al., 1998), or p21^ras-independent stimulation of Raf (Hawes et al., 1995). We compared the kinetics of p21^ras stimulation with PMA and NECA to determine whether stimulation of protein kinase C would mimic the effect of A_2B receptor activation. Our results indicate that PMA does not stimulate p21^ras within the first minute, at a time when NECA produced maximal stimulation. On the contrary, formation of the active GTP-p21^ras complexes reached a peak by 15 min after incubation with PMA, at a time when the stimulation produced by NECA was on a decline. Taken together, our data provide the first evidence that A_2B receptors can stimulate p21^ras-ERK signaling pathway in HMC-1 cells. The increase in levels of cAMP, intracellular calcium, and protein kinase C activation cannot alone explain the stimulation of p21^ras-ERK pathway produced by NECA. The events upstream of p21^ras activation remain to be delineated. Direct stimulation of p21^ras by G_q subunits is possible, as shown for angiotensin II and prostaglandin F₂ receptors (Watanabe et al., 1995; Eguchi et al., 1996).

Our study has also documented that stimulation of adenosine receptors in HMC-1 cells activates JNK and p38 MAPK cascades. This has not been previously shown for A₂ adenosine receptors. Interestingly, the time course was different for activation of these two pathways. NECA-induced activation of p38 MAPK reached its maximum within the first minute, whereas maximal activation of JNK occurred only within 10 to 15 min. The delineation of events that couple adenosine receptors with stimulation of both pathways requires further investigation.

Another novel observation was that simulation of both ERK and p38 MAPK pathways is essential for the A_2B receptor-regulated IL-8 production in human mast cells, because inhibition of either MEK, the enzyme that activates ERK, or p38 MAPK completely blocked this process. In contrast, inhibition of phosphatidylinositol 3-kinase did not affect the IL-8 production in HMC-1 cells, which is in agreement with previous observations made in mouse bone marrow mast cells (Marquardt et al., 1996). In this study we found that inhibition of protein kinase C with Ro 32-0432 completely blocked PMA-stimulated IL-8 production, but only partially blunted the response to NECA. These results suggest that stimulation of protein kinase C can contribute to IL-8 production, but this pathway alone cannot explain A_2B receptor-dependent IL-8 production.

In summary, we provide the first evidence of coupling of A₂ adenosine receptors to JNK and p38 MAPK signaling pathways, and of A_2B receptors to p21^ras- ERK activation. At least two of these MAPK pathways, namely ERK and p38 MAPK are essential for adenosine A_2B receptor-mediated production of IL-8 in the human mast cell line HMC-1. Adenosine uniquely activates mast cells in asthmatics, but not in normal persons, and the processes that explain this differential effects are unknown. The relevance of MAPKs to this phenomenon and to the pathogenesis of asthma remains to be elucidated.