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- W2014862487 abstract "Mitogen-activated protein kinase (MAPK) cascades underlie long-term mitogenic, morphogenic, and secretory activities of purinergic receptors. In HEK-293 cells,N-ethylcarboxamidoadenosine (NECA) activates endogenous A2BARs that signal through Gs and Gq/11. UTP activates P2Y2 receptors and signals only through Gq/11. The MAPK isoforms, extracellular-signal regulated kinase 1/2 (ERK), are activated by NECA and UTP. H-89 blocks ERK activation by forskolin, but weakly affects the response to NECA or UTP. ERK activation by NECA or UTP is unaffected by a tyrosine kinase inhibitor (genistein), attenuated by a phospholipase C inhibitor (U73122), and is abolished by a MEK inhibitor (PD098059) or dominant negative Ras. Inhibition of protein kinase C (PKC) by GF 109203X failed to block ERK activation by NECA or UTP, however, another PKC inhibitor, Ro 31-8220, which unlike GF 109203X, can block the ζ-isoform, and prevents UTP- but not NECA-induced ERK activation. In the presence of forskolin, Ro 31-8220 loses its ability to block UTP-stimulated ERK activation. PKA has opposing effects on B-Raf and c-Raf-1, both of which are found in HEK-293 cells. The data are explained by a model in which ERK activity is modulated by differential effects of PKC ζ and PKA on Raf isoforms. Mitogen-activated protein kinase (MAPK) cascades underlie long-term mitogenic, morphogenic, and secretory activities of purinergic receptors. In HEK-293 cells,N-ethylcarboxamidoadenosine (NECA) activates endogenous A2BARs that signal through Gs and Gq/11. UTP activates P2Y2 receptors and signals only through Gq/11. The MAPK isoforms, extracellular-signal regulated kinase 1/2 (ERK), are activated by NECA and UTP. H-89 blocks ERK activation by forskolin, but weakly affects the response to NECA or UTP. ERK activation by NECA or UTP is unaffected by a tyrosine kinase inhibitor (genistein), attenuated by a phospholipase C inhibitor (U73122), and is abolished by a MEK inhibitor (PD098059) or dominant negative Ras. Inhibition of protein kinase C (PKC) by GF 109203X failed to block ERK activation by NECA or UTP, however, another PKC inhibitor, Ro 31-8220, which unlike GF 109203X, can block the ζ-isoform, and prevents UTP- but not NECA-induced ERK activation. In the presence of forskolin, Ro 31-8220 loses its ability to block UTP-stimulated ERK activation. PKA has opposing effects on B-Raf and c-Raf-1, both of which are found in HEK-293 cells. The data are explained by a model in which ERK activity is modulated by differential effects of PKC ζ and PKA on Raf isoforms. adenosine receptor N 6-cyclopentyladenosine 2-p-(2-carboxyethyl)phenethylamino-5′-ethylcarboxaminoadenosine N 6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine 5′-N-ethylcarboxamidoadenosine 1,3-dimethylxanthine 3-propylxanthine C8-(N-methylisopropyl)-amino-N 6-(5′-endohydroxy)-endonorbornan-2-yl-9-methyladenine protein kinase A epidermal growth factor extracellular signal-regulated kinase protein kinase C 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone 2′-amino-3′-methoxyflavone 1-{6-[(17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino]hexyl}-1H-pyrrole-2,5-dione bisindolylmaleimide I 3,1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide methane sulfonate mitogen-activated protein kinase G protein-coupled receptor Diverse physiological effects of purines, adenosine, and ATP are mediated through cell surface purinergic receptors. To date, four subtypes of P1 or adenosine receptors (ARs)1 have been cloned: A1, A2A, A2B, and A3. They all belong to the G protein-coupled receptor superfamily (1Tucker A.L. Linden J. Cardiovasc. Res. 1993; 27: 62-67Crossref PubMed Scopus (202) Google Scholar). P2 (ATP) receptors are divided into two major subfamilies, the P2X receptors that are ligand-gated channels, and the P2Y receptors that are G protein-coupled (2Schachter J.B. Sromek S.M. Nicholas R.A. Harden T.K. Neuropharmacology. 1997; 36: 1181-1187Crossref PubMed Scopus (111) Google Scholar). The activation of G protein-coupled purinergic receptors has acute functional effects on all tissues that can be attributed to G protein-mediated effects on enzymes and ion channels. In addition, recent evidence indicates that purinergic receptor activation produces more slowly developing mitogenic, morphogenic, and secretory activities (3Neary J.T. Rathbone M.P. Cattabeni F. Abbracchio M.P. Burnstock G. Trends Neurosci. 1996; 19: 13-18Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 4Feoktistov I. Biaggioni I. Pharmacol. Rev. 1997; 49: 381-402PubMed Google Scholar). Recent studies have suggested that A2BARs, in addition to coupling to Gs and cyclic AMP accumulation, appear to be responsible for triggering acute Ca2+ mobilization and degranulation of canine mast cells (5Auchampach J.A. Jin J. Wan T.C. Caughey G.H. Linden J. Mol. Pharmacol. 1997; 52: 846-860Crossref PubMed Scopus (172) Google Scholar) as well as a delayed interleukin-8 release from human HMC-1 mast cells (6Feoktistov I. Biaggioni I. J. Clin. Invest. 1995; 96: 1979-1986Crossref PubMed Scopus (305) Google Scholar). A role for mast cell A2BARs in asthma is suggested by the therapeutic efficacy of theophylline and enprofylline. Both of these xanthines were found to block human A2BARs in the therapeutic dose range, and enprofylline was found to be a selective antagonist of human A2BARs (7Robeva A.S. Woodard R. Jin X. Gao Z. Bhattacharya S. Taylor H.E. Rosin D.L. Linden J. Drug Dev. Res. 1996; 39: 243-252Crossref Scopus (72) Google Scholar). Stimulation of adenylyl cyclase probably cannot account for A2BAR-mediated degranulation and stimulation of interleukin-8 synthesis from human HMC-1 mast cells, and in fact cyclic AMP has been found to be inhibitory to rodent mast cell degranulation (8Hughes P.J. Holgate S.T. Church M.K. Biochem. Pharmacol. 1984; 33: 3847-3852Crossref PubMed Scopus (154) Google Scholar, 9Hughes P.J. Church M.K. Agents & Actions. 1986; 18: 81-84Crossref PubMed Scopus (12) Google Scholar). In mast cells, activation of IgE receptors and adenosine receptors produces a synergistic interaction to trigger degranulation (10Ali H. Cunha-Melo J.R. Saul W.F. Beaven M.A. J. Biol. Chem. 1990; 265: 745-753Abstract Full Text PDF PubMed Google Scholar). IgE receptors are known to activate MAPK in mast cells (11Hirasawa N. Scharenberg A. Yamamura H. Beaven M.A. Kinet J.P. J. Biol. Chem. 1995; 270: 10960-10967Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 12Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402PubMed Google Scholar), but little is known about the regulation of this signaling pathway by adenosine receptors. The study of mast cell adenosine receptors is complicated by the fact that individual cells express multiple adenosine receptor subtypes. In addition, different adenosine receptor subtypes appear to be functionally predominant in different mast cell lines (5Auchampach J.A. Jin J. Wan T.C. Caughey G.H. Linden J. Mol. Pharmacol. 1997; 52: 846-860Crossref PubMed Scopus (172) Google Scholar, 13Hoffman H.M. Walker L.L. Marquardt D.L. Inflammation. 1997; 21: 55-68Crossref PubMed Scopus (9) Google Scholar, 14Ramkumar V. Stiles G.L. Beaven M.A. Ali H. J. Biol. Chem. 1993; 268: 16887-16890Abstract Full Text PDF PubMed Google Scholar). For this reason we decided to initially characterize functional effects of the endogenous A2BAR in HEK-293 cells where it is the only adenosine receptor expressed. ERK1/2 are 44- and 42-kDa isoform members of the MAPK family that regulate gene expression, protein synthesis, cell growth, secretion, and differentiation (15Marshall C.J. Curr. Opin. Genet. Dev. 1994; 4: 82-89Crossref PubMed Scopus (896) Google Scholar, 16Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4212) Google Scholar). MAP kinase signaling was initially shown to be activated by single-transmembrane receptor protein tyrosine kinases, such as the EGF and platelet-derived growth factor receptors. In recent years, a number of mitogenic G protein-coupled receptor (GPCR) agonists including lysophosphatidic acid (17Howe L.R. Marshall C.J. J. Biol. Chem. 1993; 268: 20717-20720Abstract Full Text PDF PubMed Google Scholar), angiotensin II (18Sadoshima J. Qiu Z. Morgan J.P. Izumo S. Circ. Res. 1995; 76: 1-15Crossref PubMed Scopus (383) Google Scholar), endothelin (19Wang Y. Pouyssegur J. Dunn M.J. J. Cardiovasc. Pharmacol. 1993; 22 Suppl. 8: S164-S167Crossref PubMed Scopus (16) Google Scholar), thromboxane A2 (20Morinelli T.A. Zhang L.M. Newman W.H. Meier K.E. J. Biol. Chem. 1994; 269: 5693-5698Abstract Full Text PDF PubMed Google Scholar), and bombesin (21Pang L. Decker S.J. Saltiel A.R. Biochem. J. 1993; 289: 283-287Crossref PubMed Scopus (71) Google Scholar) have been shown to be capable of potently activating ERK. In contrast to receptor tyrosine kinases, the intermediate steps linking GPCRs to the activation of ERK are poorly understood, and significant heterogeneity and complexity exist in the signaling pathways utilized by various GPCRs(22). It is now widely believed that the mechanism of ERK activation by GPCRs varies among cell types and individual receptors (23Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). In the present study we show that activation of HEK-293 cell adenosine receptors stimulates adenylyl cyclase, Ca2+mobilization, and ERK1/2 activation. ERK activation is Ras-dependent, but is not blocked by inhibitors of protein kinase C (PKC) or tyrosine kinases, and differs from ERK activation elicited by UTP acting on a P2Y2 receptor. We also demonstrate that A2BARs are principally responsible for initiating a sustained ERK activation in canine mastocytoma cells. CPA, NECA, CGS21680, theophylline, and enprofylline were purchased from Research Biochemicals (Natick, MA). IB-MECA was from Dr. Saul Kadin (Pfizer, Groton, CT) and WRC0571 from Dr. Pauline Martin (Discovery Therapeutics, Richmond, VA). Ro 20-1724 is from BIOMOL Research Laboratories (Plymouth Meeting, PA); phorbol 12-myristate 13-acetate, PD098059, U73122, GF 109203X, genistein, Ro 31-8220, and H-89 were from Calbiochem (San Diego, CA). A23187, pertussis toxin, and UTP from Sigma. Fura-2/AM is from Molecular Probes (Eugene, OR); adenosine deaminase from Boehringer-Mannheim; cell culture medium and LipofectAMINE were from Life Technologies, Inc. (Gaithersburg, MD). Rabbit anti-phospho-MAP kinase antibodies were raised against a synthetic peptide corresponding to the MAP kinase phosphorylation site (CTGFLT(p)EY(p)VATR) conjugated to keyhole hemocyanin (Pierce, Rockford, IL) and affinity purified negatively against the unphosphorylated peptide and positively against the phosphopeptide (24Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (962) Google Scholar). Mouse monoclonal anti-ERK2 antibody was from Upstate Biotechnology (Lake Placid, NY). Anti-B-Raf (C-19) and anti-Raf-1 (C-20) were from Santa Cruz Biotechnology (Santa Cruz, CA). Pan-Ras antibody (F111) was purchased from Santa Cruz. pcDNA3 was from Invitrogen. FLAG-tagged ERK2 was provided by Dr. S. T. Eblen. pcDNA-Ras(N17) was constructed by ligating a 0.9-kilobaseXbaI/StuI fragment from pAT-Ras(N17)(25) intoNheI/PmeI-digested pcNDA3.1(+) vector (Invitrogen, Carlsbad, CA). HEK-293 cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin at 37 °C in a humidified 5% CO2 atmosphere. Canine BR mastocytoma cells were maintained in low-glucose Dulbecco's modified Eagle's medium supplemented with 2% donor calf serum, 1.5 mml -histidine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Transient transfection of HEK-293 cells was performed on 90% confluent monolayers in 100-mm plates by means of LipofectAMINE according to the manufacturer's protocol. Empty pcDNA3 vector was added to keep the total mass of DNA added per plate constant. ERK1/2 activation assays were performed approximately 30 h after transfection. HEK-293 cells were washed twice and resuspended in serum-free Dulbecco's modified Eagle's medium/F-12 containing 15 mm HEPES, pH 7.4, 1 unit/ml adenosine deaminase, and 20 μm of the phosphodiesterase inhibitor, Ro 20-1724, and then aliquoted into test tubes. Compounds in 50-μl aliquots were added to 200 μl of cell suspension and transferred to a 37 °C shaker bath for 15 min. Assays were terminated by the addition of 500 μl of 0.15 n HCl. Cyclic AMP in the acid extract (500 μl) was acetylated and quantified by automated radioimmunoassay (26Brooker G. Terasaki W.L. Price M.G. Science. 1979; 194: 270-276Crossref Scopus (137) Google Scholar). Monolayers of HEK-293 cells were loaded with 1 μm Fura-2/AM in buffer containing 100 mm NaCl, 5 mm KCl, 1 mm MgSO4, 1 mmKH2PO4, 25 mm NaHCO3,0.5 mm CaCl2, 2.7 g/literd-glucose, 20 mm HEPES, pH 7.4, and 0.25% bovine serum albumin for 45 min. Cells were washed and resuspended in the same buffer without bovine serum albumin, plus 1 unit/ml adenosine deaminase. Fluorescence was monitored at an emission wavelength of 510 nm and excitation wavelengths of 340 and 380 nm using an SLM spectrofluorimeter in a thermostable cuvette. Prior to stimulation, HEK-293 cells or canine BR cells were serum-starved for about 18 h. Assays were carried out on monolayers of HEK-293 cells in serum-free Dulbecco's modified Eagle's medium/F-12 medium in a 37 °C, 5% CO2incubator or on suspended canine BR cells in complete Tyrode's buffer in a 37 °C shaking water bath. The reactions were terminated by placing the cells on ice and washing with ice-cold phosphate-buffered saline. The cells were then lysed in Triton lysis buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 50 mm sodium fluoride, 5 mm EDTA, 1% (v/v) Triton X-100, 40 mm β-glycerophosphate, 40 mm p-nitrophenyl phosphate, 200 μm sodium orthovanadate, 100 μm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin). The lysate was mixed and clarified by centrifugation (15 min, 14,000 rpm, 4 °C) in an Eppendorf microcentrifuge. The supernatant was subjected to SDS-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose and immunoblotting. For co-transfection experiments, FLAG-tagged ERK2 was immunoprecipitated from the cell lysate (∼400 μg) using the anti-FLAG M2 gel according to the manufacturer's instruction (Kodak) before resolution by SDS-polyacrylamide gel electrophoresis. Phosphorylation and activation of ERK1/2 was detected by immunoblotting using rabbit polyclonal anti-phospho-ERK1/2 antibody and visualized by enhanced chemiluminescence with horseradish peroxidase-conjugated goat anti-rabbit IgG as the secondary antibody (1:10,000 dilution). The membranes were then stripped by incubating in stripping buffer (62.5 mm Tris-HCl, 2% SDS, and 100 mm β-mercaptoethanol, pH 6.7, at 65 °C) in a shaking water bath, and re-probed with mouse monoclonal anti-ERK2 antibody to quantify the total ERK2 loaded onto each lane. For quantification of ERK1/2 phosphorylation, films were scanned by a laser densitometry (Molecular Dynamics) and volume integration was performed using Image QuantTM software (Molecular Dynamics). Gs-coupled A2BARs are widely expressed in tissue culture lines. To detect A2BARs in cultured HEK-293 cells, we performed cAMP accumulation assays using the non-selective adenosine receptor agonist NECA. As shown in Fig. 1 A, NECA produces a concentration-dependent increase in intracellular levels of cyclic AMP with an EC50 of 2.7 ± 0.9 μm. Fig. 1 B shows that the response to NECA (1 μm) is substantially attenuated by the A2BAR-selective antagonist enprofylline (100 μm) as well as by the non-selective AR antagonist theophylline (100 μm), but not by the A1AR-selective antagonist, WRC0571. In binding assays both enprofylline and theophylline block recombinant human A2Bwith KI values of 7 μm (7Robeva A.S. Woodard R. Jin X. Gao Z. Bhattacharya S. Taylor H.E. Rosin D.L. Linden J. Drug Dev. Res. 1996; 39: 243-252Crossref Scopus (72) Google Scholar). When added at 1 μm, CPA, IB-MECA, or CGS21680, agonists that are selective for A1, A3, A2A adenosine receptors, respectively, had little effect on intracellular cAMP in HEK-293 cells (Fig. 1 B). Only at a very high concentration (100 μm) did the A2A-selective compound CGS21680 induce a small increase in intracellular cAMP (Fig.1 B). Also, we could not detect A1, A2A, or A3 receptors by subtype-selective radioligand binding to HEK-293 cell membranes (data not shown). These findings are consistent with the observation that mRNA transcripts for A2B, but not for A1, A2A, or A3 adenosine receptor subtype have been detected in HEK-293 cells by Northern analysis (27Townsend-Nicholson A. Challiss R.A.J. Receptor Signal Transduction Protocols. Human Press, Totowa, NJ1997: 45-54Google Scholar). Collectively, these data suggest that the predominant endogenous adenosine receptors found on HEK-293 cells are A2BARs that are functionally coupled to Gsto stimulate adenylyl cyclase. We next sought to identify and characterize other signaling pathways mediated by A2BARs in HEK-293 cells. We found that NECA (1 μm) triggers transient intracellular Ca2+mobilization (Fig. 2 A), which is blocked by both enprofylline and theophylline but not by WRC0571, whereas 1 μm CPA, IB-MECA, or CGS21680 failed to provoke such a response (Fig. 2 B). Ca2+ mobilization also is elicited in response to UTP (via a P2Y2 receptor) or lysophosphatidic acid, as described in previous studies (2Schachter J.B. Sromek S.M. Nicholas R.A. Harden T.K. Neuropharmacology. 1997; 36: 1181-1187Crossref PubMed Scopus (111) Google Scholar, 28Hooks S.B. Ragan S.P. Hopper D.W. Honemann C.W. Durieux M.E. MacDonald T.L. Lynch K.R. Mol. Pharmacol. 1998; 53: 188-194Crossref PubMed Scopus (55) Google Scholar). Overnight pretreatment of HEK-293 cells with 100 ng/ml pertussis toxin had no effect on the NECA- or UTP-induced increase of intracellular Ca2+ level (data not shown), but a 15-min pretreatment with 10 μm U73122, a specific phospholipase C inhibitor (29Jin W. Lo T.M. Loh H.H. Thayer S.A. Brain Res. 1994; 642: 237-243Crossref PubMed Scopus (119) Google Scholar), completely abolished both the NECA- and UTP-induced responses (Fig.2 C). The action of NECA to increase cyclic AMP and Ca2+ signaling cannot be attributed to acute cross-talk between these two signaling pathways since forskolin elevates cyclic AMP and not Ca2+, and UTP elevates Ca2+, but not cyclic AMP (data not shown). Based on these findings, we conclude that endogenous-A2B adenosine receptors in HEK-293 cells couple to cAMP accumulation via Gs, and to Ca2+mobilization via a pertussis toxin-insensitive G protein, probably Gq/11. Many Gi-, Gq-, and some Gs-coupled receptors have been shown to elicit ERK activation in a variety of tissues and cultured cells, but little is known about the regulation of this pathway by adenosine receptors. We next set out to determine if endogenous A2BARs also couple to ERK activation in HEK-293 cells. HEK-293 cells were serum-starved overnight prior to stimulation with NECA, and ERK1/2 activation was then monitored by Western analysis using phospho-specific ERK antibodies, which only recognize activated and dually phosphorylated (Thr183 and Tyr185) ERK1/2. As shown in Fig.3, NECA evokes a time- and dose-dependent ERK1/2 activation. This activation is transient, peaks at 5 min, and gradually decreases to the baseline level in 15 min. The estimated EC50 for NECA-induced ERK1/2 activation is 0.7 μm. CPA, CGS21680, or IB-MECA (1 μm) are only weak activators of ERK1/2 compared with NECA (Fig. 4 A), and the response to NECA (0.5 μm) is blocked by enprofylline (100 μm) or theophylline (100 μm) (Fig.4 B). These data suggest that NECA-induced ERK1/2 activation in HEK-293 cells is mediated by the endogenous A2BAR.Figure 4NECA-stimulated ERK1/2 activation in HEK-293 cells is mediated by A2BARs. A, ERK1/2 activation in response to various AR agonists. Serum-starved HEK-293 cells were stimulated for 5 min with vehicle (dimethyl sulfoxide), 1 μm NECA, or 1 μm AR subtype-selective agonists: CPA (A1AR), CGS21680 (A2AAR), or IB-MECA (A3AR) prior to the determination of ERK1/2 phosphorylation. B, inhibition of NECA-stimulated ERK1/2 activation by the AR antagonists enprofylline or theophylline. Serum-starved HEK-293 cells were treated with 0.5 μm NECA in the absence or presence of 100 μm enprofylline or 100 μm theophylline prior to the determination of ERK1/2 phosphorylation. The data are normalized to the NECA-stimulated responses and each bar is the mean ± S.E. of pooled data from multiple experiments.View Large Image Figure ViewerDownload (PPT) To investigate the mechanism of A2BAR activation of ERK1/2, we first examined the effect of a highly specific inhibitor of MEK, PD098059 (30Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3241) Google Scholar). The ERK activation cascade is thought to proceed through Raf, which phosphorylates and activates MEK1/2. The MEKs phosphorylate ERK1/2 on both Thr and Tyr residues. PD098059 inhibits the activation of both MEK1 (IC50 = 5–10 μm) and MEK2 (IC50= 50 μm). As shown in Fig.5 A, ERK1/2 activation in response to 10 μm NECA stimulation was completely abolished by pretreatment for 20 min with 50 μm PD098059, suggesting that MEK1/2 are involved in A2BAR-mediated ERK1/2 activation. Next, we investigated the involvement of p21ras (Ras) in NECA-induced ERK1/2 activation. Both Ras-dependent and independent pathways have been reported for GPCR-mediated ERK activation (31Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). HEK-293 cells were transiently transfected with FLAG-tagged ERK2 together with either dominant-negative Ras-N17 or empty vector pcDNA3. Consistent with the well known involvement of Ras in receptor protein tyrosine kinase-mediated ERK activation, overexpression of Ras-N17 (confirmed by Western analysis using anti-Ras antibodies, Fig. 5 B, bottom blot) completely inhibited the ERK activation by EGF. Also inhibited were the NECA- and UTP-induced ERK activation. These data suggest that the signaling from the A2BAR or the P2Y2 receptor to ERK activation requires functional Ras in HEK-293 cells. Ras activation in response to EGF or ligands for G-protein coupled receptors such as the lysophosphatidic acid receptor in Rat-1 fibroblasts (32van Corven E.J. Hordijk P.L. Medema R.H. Bos J.L. Moolenaar W.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1257-1261Crossref PubMed Scopus (334) Google Scholar) generally requires tyrosine kinase activation, which, in most cases, can be blocked by the tyrosine kinase inhibitor, genistein. Although A2BAR and UTP receptor activation of ERK1/2 is Ras-dependent, neither response is affected by preincubation of cells with 100 μm genistein for 20 min, whereas under the same conditions, EGF-induced ERK1/2 activation was greatly reduced (Fig. 6). This suggests that NECA- and UTP-induced ERK1/2 activation in HEK-293 cells may utilize genistein-insensitive tyrosine kinases or be independent of tyrosine kinase activity. Since A2BARs are positively coupled to adenylyl cyclase, we set out to determine if increased cAMP contributes to A2BAR-induced ERK1/2 activation. Depending on the cell type, cAMP can have either a stimulatory (via B-Raf) or an inhibitory (via c-Raf-1) impact on ERK activation (33Vossler M.R. Yao H. York R.D. Pan M.G. Rim C.S. Stork P.J. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar). Western analysis reveals the presence of both B-Raf and c-Raf-1 in HEK-293 cells (data not shown). Forskolin (10 μm) increased cyclic AMP and induced a transient ERK1/2 activation in HEK-293 cells with a time course similar to that produced by NECA (data not shown). However, the magnitude of ERK activation in response to forskolin (10 μm) was about 35% lower than the activation induced by NECA (Fig. 7). The increase in intracellular cAMP in response to a 5-min simulation with forskolin (10 μm) is about 2-fold higher than that induced by NECA (10 μm, data not shown). These data indicate that cAMP accumulation can contribute to but may not fully account for the NECA-stimulated ERK activation. On the other hand, we investigated the effect of the protein kinase A inhibitor, H-89, on NECA- and forskolin-stimulated ERK activation. In a series of experiments, pretreatment of cells with H-89 (10 μm, 30 min) abolished the forskolin-induced ERK1/2 activation, whereas it only slightly decreased NECA- or UTP-induced ERK1/2 activation (Fig. 7). Taken together, these data are consistent with the hypothesis that cyclic AMP may have both stimulatory and inhibitory inputs on A2BAR-mediated ERK activation. Since A2BARs appear to couple to Gq/11 and activation of this pathway stimulates phospholipase C activity, we next set out to determine if phospholipase C is involved in NECA-stimulated ERK1/2 activation. As shown in Fig. 8, preincubation of HEK-293 cells with the specific phospholipase C inhibitor, U73122 (10 μm for 15 min), significantly attenuates (>50%) but does not eliminate NECA- and UTP-stimulated ERK1/2 activation, suggesting NECA- and UTP-induced ERK1/2 occurs at least in part via phospholipase C activation. To assess the involvement of PKC in NECA-induced ERK1/2 activation, HEK-293 cells were pretreated with the specific PKC inhibitors GF 109203X (2 μm, 15 min)(34) or Ro 31-8220 (10 μm, 15 min)(35). Whereas both inhibitors completely block phorbol 12-myristate 13-acetate-induced ERK1/2 activation, neither inhibited NECA-induced ERK1/2 activation (Fig. 9). In fact, Ro 31-8220 somewhat enhanced NECA-mediated ERK1/2 activation. UTP or the calcium ionophore A23187 induced ERK1/2 activation and showed differential sensitivity to GF 109203X and Ro 31-8220. Whereas GF 109203X had no effect on UTP or A23187-mediated ERK1/2 activation, Ro 31-8220 inhibited both. We were particularly struck by the differential effect of Ro 31-8220 on NECA- and UTP-stimulated ERK activation. Since A2BARs signal through Gs and Gq/11, and P2Y2 receptors signal through Gq/11 only, we set out to determine if the differential effect of Ro 31-8220 is due to the elevated cAMP accompanied by Gs activation. We reasoned that through simultaneous application of both UTP and forskolin to the cell, it would be possible to mimic the cellular effect of A2BAR activation by NECA. Fig. 9 C shows that in fact the combination of forskolin and UTP does mimic the NECA response and is not inhibited by Ro 31-8220. In addition, ERK activation in response to forskolin alone is enhanced by Ro 31-8220 pretreatment. These data suggest that the lack of an apparent inhibitory effect of Ro 31-8220 on A2BAR-induced ERK1/2 activation may be due to the enhancement of cyclic AMP-mediated responses by Ro 31-8220. A2BARs have recently been shown to play an important role in regulating degranulation and cytokine release from canine and human mast cells (5Auchampach J.A. Jin J. Wan T.C. Caughey G.H. Linden J. Mol. Pharmacol. 1997; 52: 846-860Crossref PubMed Scopus (172) Google Scholar). We next determined if the A2BAR-mediated ERK1/2 activation that occurs in HEK-293 can also be observed in canine BR mast cells. As shown in Fig.10, NECA elicited ERK1/2 activation in canine BR mast cells. In contrast to the transient ERK1/2 activation in HEK-293 cells, the response in canine BR cells, peaked by 1 min (the earliest time point assayed), and was sustained for at least 60 min (Fig. 10 A). This response was also completely blocked by the MEK1/2 inhibitor PD098059 (50 μm, data not shown). Compared with NECA (1 μm), CPA, IB-MECA, or CGS21680 (1 μm) were relatively weak activators of ERK1/2 in canine BR mast cells (Fig. 10 B). Furthermore, the NECA (1 μm)-induced response was blocked by enprofylline (100 μm). These data suggest that the A2BAR is principally responsible for initiating the sustained ERK1/2 activation in canine BR mast cells. The activation of purinergic receptors produce various acute G protein-mediated responses, e.g. changes in muscle tone, neuronal firing, immune function, and secretion of various hormones and cytokines. Recent studies also suggest that purines may trigger more slowly acting signal transduction cascades to mediate changes in cellular proliferation (36Sexl V. Mancusi G. Baumgartner-Parzer S. Schütz W. Freissmuth M. Br. J. Pharmacol. 1995; 114: 1577-1586Crossref PubMed Scopus (69) Google Scholar, 37Antonysamy M.A. Moticka E.J. Ramkumar V. J. Immunol. 1995; 155: 2813-2821PubMed Google Scholar), growth and differentiation (38Abbracchio M.P. Drug Dev.Res. 1996; 39: 393-406Crossref Scopus (63) Google Scholar), and apoptosis (39Tanaka Y. Yoshihara K. Tsuyuki M. Kamiya T. Exp. Cell Res. 1994; 213: 242-252Crossref PubMed Scopus (92) Google Scholar, 40Abbracchio M.P. Ceruti S. Barbieri D. Franceschi C. Malorni W. Biondo L. Burnstock G. Cattabeni F. Biochem. Biophys. Res. Commun. 1995; 213: 908-915Crossref PubMed Scopus (82) Google Scholar). MAPK cascades may regulate the latter responses. In the present study we show that stimulation of endogenous A2BARs in HEK-293 cells evokes three responses: cyclic AMP accumulation, Ca2+ mobilization, and activation of ERK1/2. This newly characterized A2BAR-mediated ERK1/2 activation and a P2Y2 receptor-mediated response elicited by UTP are dependent on Ras and MEK1/2. Both responses are attenuated by U73122, an inhibitor of phospholipase C, are completely insensitive to genistein, an inhibitor of certain tyrosine k" @default.
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- W2014862487 title "A2B Adenosine and P2Y2 Receptors Stimulate Mitogen-activated Protein Kinase in Human Embryonic Kidney-293 Cells" @default.
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