Stimulation of the Mitogen-activated Protein Kinase via the A2A-Adenosine Receptor in Primary Human Endothelial Cells*

(Received for publication, August 13, 1996, and in revised form, October 21, 1996)

Veronika Sexl Dagger , Gudrun Mancusi Dagger §, Christoph Höller , Eva Gloria-Maercker , Wolfgang Schütz and Michael Freissmuth

From the Institute of Pharmacology, University of Vienna, Währinger Straße 13a, A-1090 Vienna, Austria

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Adenosine exerts a mitogenic effect on human endothelial cells via stimulation of the A2A-adenosine receptor. This effect can also be elicited by the beta 2-adrenergic receptor but is not mimicked by elevation of intracellular cAMP levels. In the present work, we report that stimulation of the A2A-adenosine receptor and of the beta 2-adrenergic receptor activates mitogen-activated protein kinase (MAP kinase) in human endothelial cells based on the following criteria: adenosine analogues and beta -adrenergic agonists cause an (i) increase in tyrosine phosphorylation of the p42 isoform and to a lesser extent of the p44 isoform of MAP kinase and (ii) stimulate the phosphorylation of myelin basic protein by MAP kinase; (iii) this is accompanied by a redistribution of the enzyme to the perinuclear region. Pretreatment of the cells with cholera toxin (to down-regulate Gsalpha ) abolishes activation of MAP kinase by isoproterenol but not that induced by adenosine analogues. In addition, MAP kinase stimulation via the A2A-adenosine receptor is neither impaired following pretreatment of the cells with pertussis toxin (to block Gi-dependent pathways) nor affected by GF109203X (1 µM; to inhibit typical protein kinase C isoforms) nor by a monoclonal antibody, which blocks epidermal growth factor-dependent signaling. In contrast, MAP kinase activation is blocked by PD 098059, an inhibitor of MAP kinase kinase 1 (MEK1) activation, which also blunts the A2A-adenosine receptor-mediated increase in [3H]thymidine incorporation. Activation of the A2A-adenosine receptor is associated with increased levels of GTP-bound p21ras. Thus, our experiments define stimulation of MAP kinase as the candidate cellular target mediating the mitogenic action of the A2A-adenosine receptor on primary human endothelial cells; the signaling pathway operates via p21ras and MEK1 but is independent of Gi, Gs, and the typical protein kinase C isoforms. This implies an additional G protein which links this prototypical Gs-coupled receptor to the MAP kinase cascade.


INTRODUCTION

When released into or formed in the extracellular space, adenosine acts as an autacoid via interaction with four types of G protein1-coupled receptors, termed A1-, A2A-, A2B-, and A3-adenosine receptor. These receptors are encoded by distinct genes and can be differentiated based on their affinities for adenosine analogues and methylxanthine antagonists (1, 2). In addition, the receptors can be classified based on their mechanism of signal transduction; A1- and A3-adenosine receptors interact with pertussis toxin-sensitive G proteins of the Gi and Go family (3, 4, 5), whereas A2A- and A2B-adenosine receptors stimulate adenylyl cyclase via Gs (6, 7).

Adenosine is ubiquitously released by hypoxic tissues in large amounts; the nucleoside has therefore been proposed as one of the angiogenic factors that link the altered metabolism in oxygen-deprived cells to the formation of new capillaries (8). Earlier observations suggested that adenosine acts as an endothelial mitogen in vivo (9, 10). The mitogenic effect of adenosine has been verified in cultured endothelial cells derived from several vascular beds (11-13). In human endothelial cells, the proliferative response is mediated by the A2A-adenosine receptor, an effect mimicked by stimulation of the endothelial beta 2-adrenergic receptor (14). However, the mechanism by which adenosine analogues promote endothelial cell growth is not clear; there is, in particular, the apparent paradox that persistent stimulation of the signaling cascade composed of Gs, adenylyl cyclase, and protein kinase A, which is downstream of A2A-adenosine receptor, inhibits endothelial cell proliferation (14-16). In the present work, we have therefore searched for additional effector mechanisms. We report that, in human endothelial cells, the A2A-adenosine receptor stimulates the mitogen-activated protein kinase; this activation is independent of Gs, Gi, and typical protein kinase C isoforms but is associated with activation of p21ras.


EXPERIMENTAL PROCEDURES

Materials

Cell culture media were from Life Technologies, Inc., and cell culture dishes were from Greiner (Krems, Austria). [gamma -32P]ATP and [32P]orthophosphate were from DuPont NEN. Cholera toxin, CPA, (-)isoproterenol, 8-Br-cAMP, collagenase (type IV), TPA, protein A-Sepharose, rabbit anti-rat IgG, forskolin, and epidermal growth factor (EGF) were obtained from Sigma, pertussis toxin was from Peninsula Laboratories (St. Helens, UK); bFGF, guanine nucleotides, and adenosine deaminase were from Boehringer Mannheim (FRG); XAC was from Research Biochemicals (Natick, MA), GF109203X was from Calbiochem, molecular weight standards (covering the range from 14 to 97 kDa) and reagents for SDS-polyacrylamide gel electrophoresis were from Bio-Rad. PD 098059, an inhibitor of MAP kinase kinase 1 (17), was from New England BioLabs (Beverly, MA). NECA and CGS 21680 were generous gifts of Byk Gulden (Konstanz, FRG) and Ciba-Geigy (Basel, CH), respectively. Polyethyleneimine cellulose (PEI-F) TLC plates, buffers, and salts were from Merck (Darmstadt, FRG). Antibodies and antisera were obtained from the following sources: UBI (Lake Placid, NY), anti-phosphotyrosine antibody conjugated to horseradish peroxidase (16-101), rabbit antiserum directed against the p42 and p44 isoforms of MAP kinase (06-183), anti-human EGF receptor antibody (05-101), anti p21ras monoclonal rat antibody (Y13-259); New England BioLabs (Beverly, MA), rabbit antisera against the p42 and p44 isoforms of MAP kinase (9102), and the phosphorylated forms of the enzymes (9101), respectively; Sigma, rabbit anti-rat IgG; the Gsalpha -specific antiserum CS1 (18) was a generously provided by Dr. G. Milligan (Glasgow University); antiserum 7 (recognizing the G protein beta -subunits) was raised against the peptide used to obtain the original K521 antiserum (19). The immunoreactive bands on nitrocellulose blots were detected by chemiluminescence using the enhanced chemiluminescence system from Amersham (Little Chalfont, UK).

Cell Culture

Human umbilical venous endothelial cells were isolated according to Jaffe et al. (20). Cords were rinsed twice with phosphate-buffered saline and then incubated for 20 min with 0.2% collagenase in phosphate-buffered saline. The cell suspension was collected and centrifuged, and the cell pellet was resuspended in medium 199 enriched with endothelial cell growth supplement (ECGS), 100 µg/ml streptomycin, 100 units/ml penicillin, 0.25 µg/ml amphotericin B, 1 IU/ml heparin, and 20% fetal calf serum (FCS) and plated in culture dishes precoated with 1% gelatin. The cells were grown at 37 °C in a 5% CO2 humidified incubator. The following day the medium was changed to remove erythrocytes and was renewed twice a week. After 4-6 days cells formed confluent monolayers and were further subcultured, and cell plating efficiency after detachment with EDTA 0.02% (Sigma) was >98%. The thus obtained cell population consisted of >95% endothelial cells verified by their cobblestone morphology and immunofluorescence staining with antibody against von Willebrand factor antigen (Dako, Dakopatts, Denmark). Cells were used in the second and third passage.

MAP Kinase Tyrosine Phosphorylation and Immunoblots

Endothelial cells were grown to confluence on 60-mm culture dishes; subsequently, serum and growth factors were withdrawn, and the cells were maintained for 6 h in starving medium (medium 199 containing 1% methanol-extracted bovine serum albumin). Thereafter, the starving medium was replaced by medium 199 containing 1 IU/ml heparin. After an additional 30-min equilibration period, 0.1 ml of medium containing or lacking agonists and adenosine deaminase were added. Control incubations were carried out to verify that the carry-over of dimethyl sulfoxide, which resulted in maximum final concentrations of <= 0.1%, neither affected the basal level of MAP kinase phosphorylation nor the response to agonists. The incubation was carried out at 22 °C in the presence of agonists, inhibitors, and vehicle as indicated in the figure legend and terminated by rapidly rinsing (<= 10 s) with 10 ml of ice-cold phosphate-buffered saline containing 100 µM PMSF and immediately frozen in liquid N2. After thawing, the cells were scraped from the dishes in 0.1 ml of lysis buffer (in mM: 50 Tris, 40 beta -glycerophosphate, 100 NaCl, 10 EDTA, 10 p-nitrophenol phosphate, 1 PMSF, 1 Na3VO4, 10 NaF, pH adjusted to 7.4 with HCl; 1% Nonidet P-40, 0.1% SDS, 250 units/ml aprotinin, 40 µg/ml leupeptin). The unsolubilized material was removed by centrifugation at 50,000 × g for 10 min. Protein content in the lysates was measured colorimetrically using bicinchoninic acid (micro-BCA, Pierce). Aliquots corresponding to 2.5-5·104 cells (10-30 µg of protein) were dissolved in Laemmli sample buffer containing 40 mM dithiothreitol and were applied to SDS-polyacrylamide minigels (monomer concentration 12% acrylamide, 0.16% bisacrylamide, 6 cm of resolving gel). After transfer to nitrocellulose membranes, immunodetection was performed using an anti-phosphotyrosine antibody (UBI, 16-101) conjugated to horseradish peroxidase (1-2 µg/ml). Phosphorylation of MAP kinase was also assessed using an antiserum that specifically recognizes the phosphorylated p42 and p44 isoforms (New England BioLabs, 9101) at a 1:1000 dilution. Recombinant phosphorylated and nonphosphorylated p42 MAP kinase (rat extracellular signal-regulated kinase 2, New England BioLabs, 9103) were used as standards. In order to rule out that changes in immunoreactivity could simply be accounted for by different amounts of protein applied to individual lanes, the antibodies were removed by denaturation and reductive cleavage (70 °C for 30 min in 62.5 mM Tris·HCl, pH 6.8, 2% SDS, 100 mM mercaptoethanol), and the nitrocellulose blots were reprobed with an antiserum, which recognizes the p42 and p44 isoforms of MAP kinase (UBI, 06-183 or New England BioLabs, 9102) at a 1:1000 dilution. In order to detect the shift in electrophoretic mobility associated with MAP kinase activation, cellular lysates (~30-40 µg) were applied onto large SDS-polyacrylamide gels (12-cm resolving gel) containing 2 M urea.

To determine the levels of G protein subunits, endothelial cells (~5·105) were incubated in the presence of cholera toxin, forskolin, and 8-Br-cAMP as outlined in Fig. 5. After 24-48 h, the medium was removed, and the monolayer was rinsed twice with phosphate-buffered saline. The cells were detached with EDTA, resuspended in 0.5 ml of phosphate-buffered saline, and immediately frozen in liquid N2. Cell lysis was achieved by two freeze-thaw cycles. After centrifugation at 50,000 × g for 20 min, the resulting particulate fraction was dissolved in Laemmli sample buffer containing 40 mM dithiothreitol and 2% SDS; 50% aliquots were then applied to SDS-polyacrylamide gels. After transfer to nitrocellulose membranes, the splice variants of Gsalpha were visualized using the Gsalpha -specific antiserum CS1 (18) at a dilution of 1:1000. The purified recombinant splice variants of Gsalpha , rGsalpha -s, and rGsalpha -L (21) were used as standards. The remainder of the particulate fractions was used to assess the levels of the G protein beta -subunits.


Fig. 5. Phosphorylation of MAP kinase in human endothelial cells in the presence of 8-Br-cAMP and after pretreatment with cholera toxin. A, growth-arrested endothelial cells were incubated for 15 min in the presence of 2 units/ml adenosine deaminase (Ada), adenosine deaminase + 1 µM NECA (Neca); incubations in the presence of 0.5 mM 8-Br-cAMP (8Br-cAMP) were carried out in the presence of adenosine deaminase for 5, 10, and 15 min as indicated; MAP kinase phosphorylation was determined by immunoblotting with the antiserum against phospho-MAP kinase; lane Std, phosphorylated p42 MAP kinase (6 ng) was applied as a standard. B, endothelial cells were incubated for 48 h in the absence (Con) and presence of 100 ng/ml cholera toxin (Ctx), 0.5 mM 8Br-cAMP (8Br), and 10 µM forskolin (For). Particulate fractions were prepared, and 50% aliquots were subjected to immunoblotting with the Gsalpha -specific antiserum. lane Std, a mixture of purified rGsalpha -s (25 ng) and rGsalpha -L (10 ng). C and D, Endothelial cells were incubated for 36 h in the absence and presence of 100 ng/ml cholera toxin (Ctx), subsequently rendered quiescent by serum starvation (in the continuous presence of cholera toxin, where applicable) for 12 h. The stimulation was done with 1 µM NECA (Neca), 1 µM TPA (T), or 0.1 µM isoproterenol (Iso) in the presence of adenosine deaminase (Ada) for 10 min. MAP kinase phosphorylation was determined by immunoblotting with the antiserum against phospho-MAP kinase; lane Std, phosphorylated p42 MAP kinase standard (6 ng in C and 20 ng in D).
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Determination of MAP Kinase Activity

Cellular lysates from about 2 to 4·104 cells (~10-20 µg protein) were applied to SDS-polyacrylamide gels containing 0.2 mg/ml myelin basic protein (22). After electrophoresis, the gels were sequentially incubated in denaturation buffer (mM: 50 Hepes·NaOH, pH 7.4, 5 mercaptoethanol; 6 M guanidine HCl) and renaturation buffer (mM: 50 Hepes·NaOH, pH 7.4, 5 mercaptoethanol; 0.04% Tween 20), and subsequently equilibrated in kinase buffer (mM: 25 Hepes·NaOH, pH 7.4, 5 mercaptoethanol, 10 MgCl2, 0.09 Na3VO4). Renatured kinase activity was assessed by overlaying the gels with 10 ml of kinase buffer containing 250 µCi of carrier-free [gamma -32P]ATP for 30 min at 20 °C.

Immunocytochemical Staining

Endothelial cells were fixed in situ in 12-well dishes on their plastic support using acetone/methanol (1:1) or, alternatively, pure methanol at -20 °C for 20 min. After fixation, the dishes were blocked with PBS containing 3% FCS for 1 h at room temperature, incubated for 2 h with the antiserum against MAP kinase diluted in PBS containing 3% FCS, and subsequently stained with the alkaline phosphatase technique using the LSAB kit (DAKO K676) according to the instructions of the manufacturer. Briefly, cells were incubated with a biotinylated link antibody and streptavidin-coupled alkaline phosphatase; New Fuchsin was used as a chromogen.

Analysis of p21ras-bound Guanine Nucleotides

The method for determining the relative amount of GDP and GTP bound to p21ras (23) was adapted as follows. Confluent cells in 3.5-cm dishes were serum-starved for 8 h and then labeled with ~0.5 mCi of [32P]orthophosphate for an additional 16 h in serum-free, phosphate-free medium (1 ml containing 0.2% methanol-extracted BSA). The cells were washed three times with phosphate-buffered saline (prewarmed to 37 °C) and then gently overlayered with 1 ml of phosphate-free medium containing adenosine deaminase (2 units/ml) and BSA. After an additional 30-min equilibration period, agonists or vehicle (medium containing BSA) were added in 0.1 ml. After agonist stimulation, incubations were terminated by lysing the cells with ice-cold buffer (mM: 50 Tris, pH 7.5, 20 MgCl2, 150 NaCl, 1 PMSF, 1 Na3VO4; 1% Nonidet P-40, 250 units/ml aprotinin); p21ras was recovered from the lysate by immunoprecipitation with 1.5 µg of monoclonal rat antibody Y13-259 (UBI) and 10 µg of linker antibody (rabbit anti-rat IgG) complexed to protein A-Sepharose. Guanine nucleotides bound to p21ras were eluted from the immunoprecipitate by the addition of 20 µl of buffer (mM: 750 KH2PO4, pH 3.4, 5 EDTA, 1 GDP, 1 GTP) and heating (10 min at 65 °C). The eluate (10 µl/spot) was analyzed by thin layer chromatography on polyethyleneimine-coated plates (developing buffer = 1 M KH2PO4, pH 4). The radioactivity labeled and the added unlabeled nucleotides were visualized by autoradiography (Kodak X-Omat AR films, 1-4 days of exposure) and a UV lamp, respectively.

[3H]Thymidine Incorporation

The ability of bFGF, EGF, and the adenosine receptor agonist NECA to stimulate [3H]thymidine incorporation into DNA was determined as described previously (14) with minor modifications; briefly, confluent growth-arrested endothelial cells were detached with EDTA and seeded in 96-well plates (1-2·104 cells/well) in the presence of medium containing 2.5% FCS. After 5 h, an interval required for the cells to adhere, PD 098059 (final concentration = 20 µM), monoclonal anti-EGF receptor antibody (final concentration = 3 µg/ml), or vehicle was added; 1 h later, the concentration of FCS was raised to 10% (control), and the cells were incubated with the following compounds or combination of compounds at the indicated final concentrations: FCS + adenosine deaminase (2 units/ml), FCS + adenosine deaminase + NECA (1 µM), FCS + bFGF (10 ng/ml), FCS + EGF (10 ng/ml). The final volume was 0.15 ml; 15 h thereafter, 0.1 ml of medium 199 containing [3H]thymidine (0.3 to 0.5 µCi) was added for an additional 4 h. At the end on the incubation, the medium was removed, and the cells were detached by trypsinization and lysed by a freeze-thaw cycle. The particulate material was trapped onto glass fiber filters using a Skatron cell harvester, and the radioactivity retained was measured by liquid scintillation counting. The blank level of [3H]thymidine retained on the filters in the absence of DNA synthesis (determined in the presence of aphidicolin) was <50 cpm. Assays were done in sextuplicate.

Each experiment reported was carried out at least three times with three different endothelial cell batches.


RESULTS

Tyrosine Phosphorylation and Activation of MAP Kinase in the Presence of Adenosine Analogues and Isoproterenol

In order to search for potential intracellular targets downstream of the A2A-adenosine receptor, cellular lysates were obtained from endothelial cells incubated with adenosine analogues and isoproterenol; adenosine deaminase was present in the medium to remove endogenously produced adenosine. Immunoblotting with an antibody against phosphotyrosine revealed an increased immunostaining of bands migrating at 42 and 44 kDa in the presence of the nonselective adenosine analogue NECA, the highly A2A-selective adenosine analogue CGS 21680, and the beta -adrenergic agonist isoproterenol, all at 1 µM (Fig. 1A). The pattern of the other bands visualized by the phosphotyrosine antibody was not affected by the agonists employed; this also holds true for the higher and lower molecular weight range not represented in Fig. 1A. While the increase in tyrosine phosphorylation was consistently observed in the 42-kDa band, the response of the 44-kDa band was more modest (see below). In contrast to the effect of NECA and CGS 21680, the pattern of tyrosine phosphorylation was unaffected in the presence of the A1-selective agonist CPA. The phosphotyrosine-directed antibody was removed by reductive cleavage and heat denaturation, and the blots were reprobed with an antiserum directed against mitogen-activated protein kinases (MAP kinase); the 42- and 44-kDa bands, tyrosine phosphorylation of which was regulated by the agonists employed, comigrated with the p42 and p44 isoforms of MAP kinase, respectively (Fig. 1B). This suggested that A2A-adenosine receptor agonists as well as isoproterenol stimulated tyrosine phosphorylation of MAP kinases. We have verified this interpretation by using an antiserum specific for the phosphorylated forms of p42 and p44 MAP kinase (Fig. 1C). Both, the p42- and the p44-isoform of MAP kinase are present in human endothelial cells in roughly equal amounts (Fig. 1B); phosphorylation of the p42 isoform in response to mitogens was consistently observed; however, the response of the p44 isoform was modest and varied greatly in individual batches of primary cultures (cf. Figs. 1, 2, 3).


Fig. 1. Tyrosine phosphorylation and MAP kinase phosphorylation in human endothelial cells after stimulation with adenosine receptor agonists and isoproterenol. A, growth-arrested human endothelial cells were incubated for 5 min in the presence of 2 units/ml adenosine deaminase (Ada), the combination of adenosine deaminase + 0.1 µM isoproterenol (Iso), + NECA (Neca), +CPA (CPA) or +CGS 21680 (CGS), 1 µM each. Cellular lysates (20 µg) were applied onto SDS-polyacrylamide gels; immunoblotting was done with an antibody against phosphotyrosine as outlined under "Experimental Procedures." Immunodetection of all bands was uniformly suppressed in the presence of phosphotyrosine but not of phosphoserine and of phosphothreonine. B, the nitrocellulose blot shown in A was stripped and reprobed with the MAP kinase antiserum (UBI). Only the 40-kDa range of the exposure is shown, and no other immunoreactive band was detected. C, cells were incubated for 10 min in the presence of vehicle (0.1 ml control medium, lane Con.) or in the presence of adenosine deaminase and the agonists as described in A; lane Std., phosphorylated p42 MAP kinase standard (10 ng). The antiserum specific for phosphorylated MAP kinase was used for immunodetection. D, the incubation lasted 5 min in the absence (Con.) and presence of 2 units/ml adenosine deaminase (Ada), or adenosine deaminase + 0.1 µM isoproterenol (Iso), + the combination of isoproterenol and 1 µM propranolol (Prop), + 1 µM NECA (Neca), and + the combination of NECA and 10 µM xanthine amine congener (XAC). Phosphotyrosine was visualized as in A. The 40-kDa range of the exposure is shown. The double arrow indicates the position of p42 and p44 MAP kinase identified by reprobing the blots with the MAP kinase antiserum. E, assay conditions were as in D, and immunodetection was done with the antiserum against phosphorylated MAP kinase as in C; lane Std., phosphorylated p42 MAP kinase (6 ng) was applied as a standard.
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Fig. 2. Tyrosine phosphorylation and MAP kinase activation in human endothelial cells after stimulation with adenosine receptor agonists and isoproterenol. A, growth-arrested endothelial cells were incubated for 10 min in the absence (Con) and presence of 2 units/ml adenosine deaminase (Ada), adenosine deaminase + 1 µM NECA (Neca), or + 0.1 µM isoproterenol (Iso), 1 µM TPA (T), and 10 ng/ml basic fibroblast growth factor (bFGF); tyrosine phosphorylation was determined as outlined in the legend of Fig. 1 and under "Experimental Procedures." B, cellular lysates (20 µg) of endothelial cells treated as in A (a different batch of cells was used) were applied onto SDS-polyacrylamide gels containing the MAP kinase substrate myelin basic protein. Renatured kinase activity was assessed by overlaying the gels with 250 µCi of carrier-free [gamma -32P]ATP for 30 min at 20 °C. The double arrow in A and B indicates the position of p42 and p44 MAP kinase identified by reprobing the blot (A) with the MAP kinase antiserum or by immunoblotting a gel in parallel (B). C, assay conditions were as outlined in A. Cellular lysates (40 µg) were applied onto a SDS-polyacrylamide gel with a resolving distance of 12 cm containing 2 M urea to detect the reduced electrophoretic mobility associated with phosphorylation of MAP kinase. Immunoblotting was done with an antiserum against p42 and p44 MAP kinase (New England BioLabs).
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Fig. 3. Time course of tyrosine phosphorylation and MAP kinase phosphorylation in human endothelial cells after stimulation with NECA and isoproterenol. A, growth-arrested endothelial cells were incubated in the presence of 2 units/ml adenosine deaminase (Ada) with 0.1 µM isoproterenol (Iso) or 1 µM NECA (Neca) for 3, 5, 10, and 20 min as indicated, and the level of tyrosine phosphorylation was determined by immunoblotting with the phosphotyrosine antibody. The control incubation containing adenosine deaminase (Ada) lasted 20 min. B, the nitrocellulose blot shown in A was stripped and reprobed with the MAP kinase antiserum to verify that the changes visualized in A cannot be accounted for by variabilities in the amount of cellular proteins applied to the gel. C, assay conditions were as outlined in A; lane Con, addition of 0.1 ml of medium followed by a 20-min control incubation; lane Std, phosphorylated p42 MAP kinase (6 ng) was applied as a standard. Only the 40-kDa range of the exposures is shown.
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Incubation of the cells with the adenosine receptor antagonist XAC completely prevented the NECA-induced increase in MAP kinase phosphorylation irrespective of whether immunodetection was performed with the anti-phosphotyrosine antibody (Fig. 1D) or with the antiserum against phosphorylated MAP kinase (Fig. 1E). Similarly, the beta -adrenergic antagonist L-propranolol blocked the isoproterenol-induced response (Fig. 1, D and E).

We have compared the ability of NECA and isoproterenol to promote tyrosine phosphorylation of the 42-kDa band with that of basic fibroblast growth factor and the phorbol ester TPA (1 µM); both compounds are potent and efficient mitogens for human endothelial cells. In most cell batches, incubation of the endothelial cells with the latter two compounds resulted in higher levels of phosphotyrosine incorporation into the 42- and 44-kDa bands than NECA or isoproterenol (Fig. 2A). This is consistent with the efficiency with which the compounds stimulate proliferation; under our culture conditions, TPA and bFGF are the strongest mitogenic stimuli.

We have verified that the increase in tyrosine phosphorylation resulted in higher MAP kinase activity. Lysates from control and stimulated cells were applied to a polyacrylamide gel containing the MAP kinase substrate myelin basic protein. Following renaturation, MAP kinase activity was detected in the presence of [gamma -32P]ATP. An increase of myelin basic protein phosphorylation was observed in the 42-/44-kDa region of the gel in response to bFGF, TPA, NECA, and isoproterenol (Fig. 2B). Phosphorylation of MAP kinases slows their migration through polyacrylamide gels. This was not seen with the minigel system (6 cm resolving gel) employed to generate the data depicted in Fig. 1. However, the shift to slower mobility was detectable if a larger gel was employed (Fig. 2C). The isoproterenol- and NECA-induced stimulation of tyrosine phosphorylation of MAP kinase gradually increased, the maximum being reached after 10-15 min (Fig. 3, A and C) and maintained for at least 50 min (not shown).

Translocation of MAP Kinase

Persistent activation of MAP kinase results in cellular redistribution such that a fraction migrates into the nucleus, where it phosphorylates target proteins (24-26). We have determined the cellular localization of MAP kinases by immunocytochemistry in order to search for an agonist-induced redistribution. In untreated control cells, immunoreactive material was primarily observed in the cellular periphery yielding a ring-like staining pattern (Fig. 4A). If the endothelial cells were incubated with the phorbol ester TPA (1 µM), the immunoreactive material was found almost exclusively in the perinuclear region (Fig. 4C). Addition of NECA (Fig. 4B) or of isoproterenol (not shown) altered the distribution of the immunoreactive material in a similar manner, although the effect was less pronounced than that of TPA. The different extent of translocation correlates with the efficiency with which the compounds stimulate proliferation (see above).


Fig. 4. Immunocytochemical staining of MAP kinase in human endothelial cells. Growth-arrested human endothelial cells were pretreated with adenosine deaminase (A). Stimulation was carried out with 1 µM NECA (B) or 1 µM TPA (C) for 20 min. Cells were subsequently fixed with acetone/methanol (1:1) at -20 °C for 20 min; staining with the MAP kinase antibody, biotinylated link antibody, and streptavidin-coupled alkaline phosphatase was carried out as described under "Experimental Procedures."
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Delineation of the G Protein-dependent Signaling Cascade

The A2A-adenosine and the beta -adrenergic receptors are coupled to adenylyl cyclase activation via Gs. However, if the cAMP/protein kinase A signaling cascade was directly activated by incubating the cells with a high concentration of the membrane-permeable cAMP analogue 8Br-cAMP, no increase in the phosphorylation of the p42 and p44 MAP kinase was seen when compared with the reference incubation in the presence of adenosine deaminase or to NECA-stimulated cells (Fig. 5A); similar results were obtained if the levels of phosphotyrosine were determined (not shown). Thus, cAMP generated by A2A-adenosine receptor-mediated adenylyl cyclase stimulation is unlikely to account for activation of MAP kinase. In order to determine whether another Gs-regulated effector was involved in the activation of MAP kinase, we have exploited the fact that persistent activation of Gsalpha via cholera toxin but not of the downstream signaling cascade cells leads to its down-regulation (18, 27). This phenomenon was also seen in endothelial cells treated for 48 h with cholera toxin (Fig. 5B); an essentially complete down-regulation of both splice variants of Gsalpha was already observed after a 24-h incubation with cholera toxin, whereas the levels of G protein beta -subunits were unaffected (not shown). In spite of the profound reduction in the levels of Gsalpha , activation of the A2A-adenosine receptor by NECA still promoted the phosphorylation of MAP kinase (Fig. 5, C and D). In some, but not all cell preparations, the effect of NECA appeared to be augmented by cholera toxin pretreatment (cf. Fig. 5, C and D). In contrast, cholera toxin pretreatment essentially abolished the response to isoproterenol (Fig. 5D). This indicates that the beta 2-adrenergic receptor but not the A2A-adenosine receptor requires functional Gsalpha for MAP kinase activation.

Under appropriate conditions, seven transmembrane spanning receptors can interact with several distinct classes of G proteins. Gialpha -2 is the most abundant G protein alpha -subunit in endothelial membranes and is required to support endothelial cell growth in the presence of serum-derived growth factors (14). We have therefore pretreated the endothelial cells with pertussis toxin to block the functional coupling between receptors and Gi. This preincubation eliminates the functional response to Gi-dependent endothelial mitogens present in FCS (14). However, phosphorylation of MAP kinase in response to activation of the A2A-adenosine receptor was not reduced but rather enhanced following pertussis toxin-pretreatment (Fig. 6A).


Fig. 6. Phosphorylation of MAP kinase in human endothelial cells after pretreatment with pertussis toxin and in the presence of GF109203X. A, endothelial cells were growth-arrested by serum deprivation in the absence and presence of 100 ng/ml pertussis toxin for 24 h. Subsequently, the incubation was done for 10 min in the presence of 2 units/ml adenosine deaminase in the absence (Ada) and presence of 1 µM NECA (Neca), 0.1 µM isoproterenol (Iso), or 1 µM TPA (T); lane Std, phosphorylated p42 MAP kinase standard (6 ng). B, growth-arrested human endothelial cells were incubated for 30 min in the absence and presence of 1 µM GF109203X (GF 1 µM) and subsequently stimulated for 15 min in the presence of 2 units/ml adenosine deaminase (A) with 0.1 µM isoproterenol (Iso), or 1 µM NECA (Neca), 1 µM CGS 21680 (CGS), 1 µM TPA (T). Control incubations (Co) were also done in the absence of adenosine deaminase. MAP kinase phosphorylation in A and B was determined by immunoblotting with the antiserum against phospho-MAP kinase.
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Direct stimulation of protein kinase C isoforms by the phorbol ester TPA results in MAP kinase stimulation in endothelial cells (cf. Figs. 2 and 4). The phospholipase Cbeta /protein kinase C signaling cascade is subject to two types of regulation by G protein subunits. Free G protein beta gamma -dimers are generated by subunit dissociation from G proteins of the Gi/Go class, which in most cells account for the bulk of the G proteins, and thus mediates pertussis toxin-sensitive stimulation of phospholipase Cbeta . The second pathway involves G protein alpha -subunits of the Gq family. We have therefore also determined the effect of the protein kinase C inhibitor GF109203X on tyrosine phosphorylation of MAP kinase in response to A2A-adenosine and beta 2-adrenergic receptor stimulation. At 1 µM, GF109203X inhibited MAP kinase activation induced by the phorbol ester TPA but did not interfere with the NECA- and isoproterenol-induced tyrosine phosphorylation (Fig. 6B). Identical results were obtained, if the cells were pretreated with the phorbol ester TPA for 24 h which resulted in down-regulation of the typical protein kinase C isoforms alpha  and beta  and to a lesser extent of protein kinase Cepsilon . Raising the concentration of GF109203X to 30 µM blocked the effect of NECA and isoproterenol; under these conditions, however, the level of basal tyrosine phosphorylation was also reduced indicating that the concentration range at which GF109203X was selective had been exceeded (data not shown).

Tyrosine kinases activate MAP kinase via a signaling cascade that results in guanine nucleotide exchange on p21ras and subsequent stimulation of raf kinase activity. G protein-coupled receptors are also capable of activating p21ras in several cell lines (28, 29). We have therefore stimulated endothelial cells with bFGF and the adenosine receptor agonist NECA and determined the level of GDP- and GTP-bound p21ras. Under basal conditions, determined in the presence of adenosine deaminase, the immunoprecipitate contained predominantly radiolabeled GDP (Fig. 7). Both, NECA and bFGF, caused a time-dependent increase in the level of GTP bound to p21ras (Fig. 7).


Fig. 7. Activation of p21ras in human endothelial cells. Growth-arrested human endothelial cells were metabolically labeled with [32P]orthophosphate for 16 h and subsequently equilibrated in medium containing 2 units/ml adenosine deaminase for 30 min. Thereafter (time = 0), NECA and bFGF were added to a final concentration of 1 µM and 10 ng/ml, respectively; an equivalent volume of vehicle (0.1 ml of medium containing BSA) was added to the control incubation (Ada). The incubation continued for 2, 5, and 10 min, as indicated. The cells were lysed, and p21ras was recovered from the lysates by immunoprecipitation, and guanine nucleotides bound to p21ras were analyzed by thin layer chromatography as described under "Experimental Procedures."
[View Larger Version of this Image (59K GIF file)]


In p21ras-dependent mitogenic signaling the activation of the raf kinase by p21ras is linked to stimulation of MAP kinase via MAP kinase kinase (MEK). To confirm that the A2A-adenosine receptor-induced activation of p21ras and MAP kinase were linked, we have used the inhibitor PD 098059, which blocks the activation of MEK1 (17). Preincubation of endothelial cells with PD 098059 blocked the NECA-induced stimulation of MAP kinase and greatly reduced the response to bFGF (Fig. 8A). Likewise, the inhibitor also abolished the effect of isoproterenol on MAP kinase (not shown). At the concentration employed (20 µM), PD 098059 is selective for MEK1 (IC50 = 2-7 µM, Ref. 17), whereas higher concentrations are required to block activation of MEK2 (IC50 = 50 µM, Ref. 17).


Fig. 8. Phosphorylation of MAP kinase in human endothelial cells after pretreatment with PD 098059 (A) and a monoclonal antibody against the human EGF receptor (B). Growth-arrested human endothelial cells incubated for 60 min in the absence and presence of 20 µM PD 098059 (A, PD) or 3 µg/ml monoclonal antibody (mAb) against the human EGF receptor (B, mAb) and subsequently stimulated for 15 min in presence of 2 units/ml adenosine deaminase (Ada) with 1 µM NECA (Neca), with 10 ng/ml bFGF (FGF), or 10 and 100 ng/ml EGF (EGF) as indicated; lane Std, phosphorylated p42 MAP kinase standard (6 ng). MAP kinase phosphorylation in A and B was determined by immunoblotting with the antiserum against phospho-MAP kinase.
[View Larger Version of this Image (37K GIF file)]


If the A2A-adenosine receptor-induced mitogenic response and activation MAP kinase were causally related, PD 098059 ought to block the ability of NECA (and of bFGF) to stimulate DNA synthesis. This was the case (Table I). In the absence of PD 098059, bFGF and NECA stimulated [3H]thymidine incorporation ~1.7- and ~1.6-fold over the respective control incubations (FCS and FCS + adenosine deaminase). Preincubation of the cells with 20 µM PD 098059 did not affect cell viability but reduced the levels of [3H]thymidine incorporation observed in the presence of FCS and of FCS + adenosine deaminase. More importantly, in the presence of PD 098059, the response to bFGF was clearly blunted (1.2-fold stimulation), and the effect of NECA was essentially undetectable (1.1-fold stimulation).

Table I.

Stimulation of [3H]thymidine incorporation by NECA, bFGF, and EGF in the absence and presence of PD 098059 and of a monoclonal antibody against the human EGF receptor

[3H]Thymidine incorporation was determined as outlined under "Experimental Procedures": the final concentrations were 10% FCS, 2 units/ml adenosine deaminase. 1 µM NECA, 10 ng/ml EGF, 10 ng/ml bFGF, 20 µM PD 098059, and 3 µg/ml monoclonal antibody against the human EGF receptor (=anti-EGFRmAb). The levels of [3H]thymidine incorporation determined in the presence of 10% FCS were set 100% to normalize for intra assay variations. The absolute values were between 1500 and 3500 cpm in individual experiments; this variation was attributable in part to the different number of cells seeded per well (1-2 · 104) in each experiment. Data are means ± S.D. from three independent experiments which were carried out in sextuplicate; ND, not determined.
FCS FCS + bFGF FCS + EGF FCS + adenosine deaminase FCS + adenosine deaminase + NECA

% % % % %
No addition 100 173  ± 12 167  ± 20 83  ± 9 134  ± 10
+PD 098059 77  ± 11 94  ± 17 ND 68  ± 13 77  ± 16
+Anti-EGFRmAb 106  ± 9 ND 118  ± 8 96  ± 6 163  ± 19

Recent experiments in rat-1 fibroblasts indicate that activation of MAP kinase by G protein-coupled receptors depends on a mechanism that required a functional EGF receptor (30). As mentioned earlier, we did not observe any increase in tyrosine phosphorylation in the high molecular weight range of the immunoblots shown in Fig. 1. This, however, may have escaped detection because of an unfavorable signal-to-noise ratio. We have therefore used two approaches to assess the contribution of EGF receptor-dependent pathways to the A2A-adenosine receptor-induced response. (i) The endothelial cells were preincubated with several tyrosine kinase inhibitors such as genistein, tyrphostin 25, tyrphostin 23, and tyrphostin 51 for up to 2 h. None of these compounds inhibited MAP kinase activation by NECA up to concentrations of 50 µM. However, these results were inconclusive since the inhibitors also failed to block the response elicited by bFGF and EGF. This suggested that human endothelial cells did not accumulate significant amounts of these compounds (data not shown). (ii) Alternatively, cells were preincubated with a monoclonal antibody capable of blocking the human EGF receptor. At a concentration of antibody that blocked the increase in MAP kinase phosphorylation induced by 10 ng/ml EGF and substantially reduced the effect of 10-fold higher EGF concentration, no inhibition of the NECA-induced response was seen (Fig. 8B). Similarly, the EGF-induced stimulation of [3H]thymidine incorporation (~1.7-fold over the FCS control, see Table I) was essentially eliminated by the antibody (~1.1-fold of the reference value = FCS + antibody, see Table I). In contrast the stimulation by NECA remained unaffected by the antibody (~1.6- and 1.7-fold stimulation over the corresponding reference values, i.e. FCS + adenosine deaminase in the absence and presence of antibody, respectively, see Table I).


DISCUSSION

In the present work, we show that the A2A-adenosine receptor stimulates MAP kinase (predominantly the p42 isoform) in primary cell cultures of human endothelial cells. Our experiments have identified MAP kinase as an intracellular target, which can account for the growth-stimulating activity of adenosine on endothelial cells, with p21ras and MAP kinase kinase 1 as components of the underlying signaling pathway.

MAP kinases or extracellular signal-regulated kinases are rapidly stimulated by growth promoting factors acting on a variety of cell surface receptors (24). In turn, MAP kinases phosphorylate and regulate key intracellular enzymes and transcription factors required for G0/G1 transition and cellular proliferation. Receptors with tyrosine kinase activity activate MAP kinases in a multistep process that involves a limited number of defined protein-protein interactions, guanine nucleotide exchange on p21ras, and activation of a downstream protein kinase cascade (24-26). In contrast, MAP kinase activation by G protein-coupled receptors is less well understood. At least five distinct pathways for G protein-mediated activation of MAP kinase have been documented. (i) Stimulation of Gi-coupled receptors results in the formation of GTP-bound p21ras in fibroblast cell lines (28, 29). In most cells, pertussis toxin-sensitive G proteins of the Gi/Go family are the most abundant such that receptor-induced G protein subunit dissociation will generate high levels of free beta gamma -dimers in the membrane. Activation of p21ras may actually be accomplished by the beta gamma -dimers. Overexpression of beta gamma -subunits rather than alpha -subunits is associated with increased p21ras-dependent signaling (31); this effect can be reversed by the beta gamma -dimer binding fragment of the beta -adrenergic receptor kinase 1/G protein-coupled receptor kinase 2 (32) and is mediated by tyrosine kinase-dependent phosphorylation of Shc adapter proteins (33). (ii) MAP kinase activation can also be achieved through G protein-coupled receptors via protein kinase C isoforms in response to increased diacylglycerol levels generated from phospholipid precursors by phospholipase C or phospholipase D (25, 34). (iii) Receptor-promoted calcium entry (22, 35) and (iv) elevation of cAMP may lead to MAP kinase activation in some cells (34, 36). (v) Recently, MAP kinase activation by G protein-coupled receptors (for lysophosphatidic acid, thrombin, and endothelin-1) has been shown to depend on tyrosine phosphorylation of the EGF receptor in rat-1 fibroblasts. This phenomenon has been termed "transactivation" of the EGF receptor (30). Our observations rule out several of these mechanisms in linking the A2A-adenosine receptor to MAP kinase activation. In particular, raising intracellular cAMP in endothelial cells does not per se induce tyrosine phosphorylation of MAP kinase. Similarly, activation of protein kinase C isoforms via a Gq-dependent stimulation of phospholipase Cbeta is not involved as the underlying mechanism; the protein kinase C inhibitor GF109203X is inactive at concentrations that essentially eliminate the activation of MAP kinase by the phorbol ester TPA. The ability of GF109203X to block the NECA-induced MAP kinase activation at high concentrations most likely reflects loss of selectivity and is thus impossible to interpret. We have also failed to detect calcium transients in endothelial cells loaded with the indicator dye Fura-2 after addition of NECA or isoproterenol, although transient increases in intracellular calcium were seen in response to activation of the Gq-coupled H1-histamine receptor.2 Finally, a functional EGF receptor is not required for activation of MAP kinase via the A2A-adenosine receptor. Obviously, we cannot rule out that a receptor tyrosine kinase other than the EGF receptor is transactivated by the A2A-adenosine receptor in endothelial cells. This type of cooperation between tyrosine kinase receptors and G protein-coupled receptors may be a general phenomenon and specific for cell types or receptor pairs, e.g. in rat vascular smooth muscle cells, stimulation of the angiotensin II type 1 receptor causes tyrosine phosphorylation and activation of the platelet-derived growth factor receptor (37). In contrast, transactivation of the platelet-derived growth factor receptor is not seen in rat-1 fibroblasts (28).

Both the A2A-adenosine receptor and the beta 2-adrenergic receptor are prototypical Gs-coupled receptors. Endothelial cells express beta 2-adrenergic receptors (38); although it is doubtful that epinephrine and/or norepinephrine participate in the regulation of angiogenesis in vivo, beta -adrenergic agonists exert a mitogenic effect in cultured endothelial cells (14). Our observations show that A2A-adenosine receptor and beta 2-adrenergic receptor use distinct mechanisms to activate MAP kinase in endothelial cells. Tyrosine phosphorylation of MAP kinase in response to beta 2-adrenergic receptor activation is abolished after cholera toxin pretreatment; in contrast, the A2A-adenosine receptor does not depend on Gsalpha to stimulate MAP kinase. Hence, we conclude that, in endothelial cells, the signaling pathways by which the two receptors impinge on the MAP kinase cascade require different G proteins.

So far, no interaction of the A2A-adenosine receptor with G proteins other than Gs has been reported; in turkey erythrocytes beta -adrenergic receptors stimulate phospholipase Cbeta via Gq (39, 40), an effect which cannot be mimicked by the A2A-adenosine receptor (40). Gs-coupled receptor such as the beta 2-adrenergic receptor and the TSH receptor have also been shown to interact with the G12/G13 protein class (41, 42) and to thereby activate the Na+/H+-antiporter (43). Galpha 12 and Galpha 13 are expressed in the human endothelial cells used in the present study, and the stimulation of the A2A-adenosine receptor can stimulate intracellular alkalinization which is blocked by the Na+/H+-exchange inhibitor HOE694 (data not shown); thus, the endothelial A2A-adenosine receptor may couple directly to G12 and/or G13. In addition, beta -adrenergic receptors are known to directly interact with Gi under appropriate conditions (44). In endothelial cells, Gi is clearly not involved in MAP kinase activation by A2A-adenosine and beta 2-adrenergic receptors since pertussis toxin failed to block the NECA- and isoproterenol-induced response. Surprisingly, preincubation with pertussis toxin augmented the response to NECA. This was also seen in some cell batches following cholera toxin pretreatment. We currently cannot provide a mechanistic explanation for this phenomenon. However, we have recently observed that, in sympathetic neurons, down-regulation of Gsalpha causes sensitization of the alpha 2-adrenergic autoreceptors that signal via alpha -subunits of the Gi/Go subfamily (45). This suggests that changing the availablility of functional G protein alpha -subunits may also affect the efficiency of signal transfer through pathways that are controlled by unrelated G proteins.

To our knowledge, there are only two other cell types in which a growth-promoting effect of the A2A-adenosine receptor has been documented, namely NIH3T3 cells and thyroid epithelial cells. The ability of adenosine analogues to enhance the proliferation of NIH3T3 cells was thought to arise from elevation of intracellular cAMP levels that support the growth induced by obligatory mitogenic signals such as activation of tyrosine kinase-coupled receptors (46). When placed under the control of the thyroglobulin promoter and expressed ectopically as a transgene in the thyroid gland, the A2A-adenosine receptor is capable of inducing large hyperfunctioning goiters (47); this was interpreted as evidence for continuous activation of the cAMP-signaling cascade which stimulated thyroid growth such that adenosine substituted for TSH, the physiological regulator of the thyroid. However, in primary cultures of human thyroid epithelial cells, activation of the TSH receptor, a Gs-coupled receptor which, nevertheless, has the capacity to activate essentially all G protein alpha -subunits (42) leads to tyrosine phosphorylation of the p42 isoform of MAP kinase, and this effect is neither mimicked by activation of the Gs/adenylyl cyclase/protein kinase A cascade nor blocked by pertussis toxin treatment (48). Thus, the TSH receptor and the A2A-adenosine receptor may share a common signaling mechanism that links them to MAP kinase activation in the appropriate cellular background. In human endothelial cells, stimulation of guanine nucleotide exchange on p21ras and subsequent activation of MEK1 are part of the intervening signaling cascade.


FOOTNOTES

*   This work was supported by Grant P10672-MED from the Austrian Science Foundation (to M. F.) and from the Science Foundation of the Vienna City Council (to V. S.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Contributed equally to this work.
§   Present address: University Clinic of Otorhinolaryngology, Vienna General Hospital, Währinger Gürtel 18-20; A-1090 Vienna; Austria.
   To whom correspondence should be addressed: Institute of Pharmacology, Vienna University; Währinger Str. 13a, A-1090 Vienna; Austria. Tel.: 43-1-40 480/298; Fax: 43-1-402 48 33; E-mail: michael.freissmuth{at}univie.ac.at.
1    The abbreviations used are: G protein, regulatory GTP-binding protein; MAP kinase, mitogen-activated protein kinase; BSA, bovine serum albumin; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; FCS, fetal calf serum; NECA, 5'-N-ethylcarboxamidoadenosine; CGS 21680, 5'-N-ethylcarboxamido-2-[4-(2-carboxyethyl)phenylethyl]adenosine; CPA, N6-cyclopentyladenosine; XAC, xanthine amine congener; 8Br-cAMP, 8-bromocyclic AMP, TPA, 12-O-tetradecanoylphorbol-13-acetate; GF109203X, bisindoylmaleimide I; PMSF, phenylmethylsulfonyl fluoride; MEK, MAP kinase kinase.
2    S. Boehm and V. Sexl, unpublished observations.

Acknowledgments

We are grateful to Dr. G. Milligan for a gift of antiserum CS1 and to Drs. M. Baccharini, C. Nanoff, F. Überall, and L. Wagner for support and advice. We thank A. Karel and E. Tuisl for technical assistance and for artwork, respectively. We also thank the midwives and nurses and the newborn babies of the Department of Gynecology and Obstetrics, Vienna General Hospital, for providing us with umbilical cords.


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