(Received for publication, August 13, 1996, and in revised form, October 21, 1996)
From the Institute of Pharmacology, University of Vienna, Währinger Straße 13a, A-1090 Vienna, Austria
Adenosine exerts a mitogenic effect on human
endothelial cells via stimulation of the A2A-adenosine
receptor. This effect can also be elicited by the
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
2-adrenergic receptor activates mitogen-activated protein kinase (MAP kinase) in human endothelial cells based on the
following criteria: adenosine analogues and
-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 Gs
) 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.
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 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.
Cell culture media were from Life Technologies,
Inc., and cell culture dishes were from Greiner (Krems, Austria).
[-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
Gs
-specific antiserum CS1 (18) was a generously provided
by Dr. G. Milligan (Glasgow University); antiserum 7 (recognizing the G
protein
-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).
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 ImmunoblotsEndothelial 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
-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
Gs were visualized using the Gs
-specific
antiserum CS1 (18) at a dilution of 1:1000. The purified recombinant
splice variants of Gs
, rGs
-s,
and rGs
-L (21) were used as standards. The remainder of the particulate fractions was used to assess the levels of
the G protein
-subunits.
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
[-32P]ATP for 30 min at 20 °C.
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.
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 IncorporationThe 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.
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 -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).
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 -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 [-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).
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).
Delineation of the G Protein-dependent Signaling Cascade
The A2A-adenosine and the -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 Gs
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 Gs
was already observed after a 24-h
incubation with cholera toxin, whereas the levels of G protein
-subunits were unaffected (not shown). In spite of the profound
reduction in the levels of Gs
, 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
2-adrenergic
receptor but not the A2A-adenosine receptor requires
functional Gs
for MAP kinase activation.
Under appropriate conditions, seven transmembrane spanning receptors
can interact with several distinct classes of G proteins. Gi-2 is the most abundant G protein
-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).
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 C/protein kinase C
signaling cascade is subject to two types of regulation by G protein
subunits. Free G protein
-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
C
. The second pathway involves G protein
-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
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
and
and to a lesser
extent of protein kinase C
. 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).
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).
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).
|
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).
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 -dimers in the membrane. Activation of
p21ras may actually be accomplished by the
-dimers.
Overexpression of
-subunits rather than
-subunits is
associated with increased p21ras-dependent
signaling (31); this effect can be reversed by the
-dimer binding
fragment of the
-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 C
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
2-adrenergic receptor are prototypical
Gs-coupled receptors. Endothelial cells express
2-adrenergic receptors (38); although it is doubtful
that epinephrine and/or norepinephrine participate in the regulation of
angiogenesis in vivo,
-adrenergic agonists exert a
mitogenic effect in cultured endothelial cells (14). Our observations
show that A2A-adenosine receptor and
2-adrenergic receptor use distinct mechanisms to activate MAP kinase in endothelial cells. Tyrosine phosphorylation of
MAP kinase in response to
2-adrenergic receptor
activation is abolished after cholera toxin pretreatment; in contrast,
the A2A-adenosine receptor does not depend on
Gs
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 -adrenergic receptors stimulate phospholipase C
via
Gq (39, 40), an effect which cannot be mimicked by the
A2A-adenosine receptor (40). Gs-coupled
receptor such as the
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).
G
12 and G
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,
-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
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 Gs
causes sensitization of the
2-adrenergic autoreceptors that signal via
-subunits
of the Gi/Go subfamily (45). This suggests that
changing the availablility of functional G protein
-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 -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.
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.