From the Departments of Molecular Physiology and
Biological Physics, § Microbiology and ¶ Internal
Medicine, University of Virginia,
Charlottesville, Virginia 22908
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ABSTRACT |
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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 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 (1).
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 (2). 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 (3, 4).
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 (5) as well as a delayed
interleukin-8 release from human HMC-1 mast cells (6). 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 (7). 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
(8, 9). In mast cells, activation of IgE receptors and adenosine
receptors produces a synergistic interaction to trigger degranulation
(10). IgE receptors are known to activate MAPK in mast cells (11, 12),
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 (5, 13, 14). 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 (15, 16). 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 (17), angiotensin II
(18), endothelin (19), thromboxane A2 (20), and bombesin
(21) 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 (23).
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.
Materials--
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 (24). 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-kilobase
XbaI/StuI fragment from pAT-Ras(N17)(25) into
NheI/PmeI-digested pcNDA3.1(+) vector
(Invitrogen, Carlsbad, CA).
Cell Culture and Transfection--
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 mM
L-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.
Cyclic AMP Assays--
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 (26).
Measurement of Intracellular Ca2+--
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 mM
KH2PO4, 25 mM NaHCO3,
0.5 mM CaCl2, 2.7 g/liter
D-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.
ERK1/2 Activation Assay--
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% CO2
incubator 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 Endogenous A2BARs Evoke cAMP Accumulation and Ca2+
Mobilization in HEK-293 Cells--
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. 1A,
NECA produces a concentration-dependent increase in
intracellular levels of cyclic AMP with an EC50 of 2.7 ± 0.9 µM. Fig. 1B 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 A2B
with KI values of 7 µM (7). 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. 1B). Only at a very high concentration
(100 µM) did the A2A-selective compound
CGS21680 induce a small increase in intracellular cAMP (Fig.
1B). 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 (27). Collectively, these data suggest that
the predominant endogenous adenosine receptors found on HEK-293 cells
are A2BARs that are functionally coupled to Gs
to 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. 2A), 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. 2B). Ca2+ mobilization
also is elicited in response to UTP (via a P2Y2 receptor)
or lysophosphatidic acid, as described in previous studies (2, 28).
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 (29),
completely abolished both the NECA- and UTP-induced responses (Fig.
2C). 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.
Endogenous A2BARs Activate ERK1/2 in HEK-293
Cells--
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. 4A), and the response to
NECA (0.5 µM) is blocked by enprofylline (100 µM) or theophylline (100 µM) (Fig.
4B). These data suggest that NECA-induced ERK1/2 activation
in HEK-293 cells is mediated by the endogenous A2BAR.
A2BAR-induced ERK1/2 Activation Is MEK1/2- and
Ras-dependent--
To investigate the mechanism of
A2BAR activation of ERK1/2, we first examined the effect of
a highly specific inhibitor of MEK, PD098059 (30). 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.
5A, 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 (31). 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. 5B, 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.
Insensitivity of A2BAR-mediated ERK1/2 Activation to the
Tyrosine Kinase Inhibitor Genistein--
Ras activation in response to
EGF or ligands for G-protein coupled receptors such as the
lysophosphatidic acid receptor in Rat-1 fibroblasts (32) 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.
Effect of Elevated Intracellular Cyclic AMP on
A2BAR-mediated ERK1/2 Activation--
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 (33). 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.
Effect of the Phospholipase C Inhibitor U73122 on
A2BAR-mediated ERK1/2 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.
Differential Effects of PKC Inhibitors on A2BAR- and
UTP-mediated ERK1/2 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. 9C 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 Initiate Sustained ERK1/2 Activation in Canine BR
Mast Cells--
A2BARs have recently been shown to play an
important role in regulating degranulation and cytokine release from
canine and human mast cells (5). 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. 10A). 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. 10B). 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 (36, 37), growth and differentiation (38), and
apoptosis (39, 40). 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 kinases, and are only
minimally affected by the PKA inhibitor, H-89. In this study, we have
demonstrated for the first time the existence of an interesting
interaction between PKA and PKC in regulating ERK activity by
endogenous GPCRs in HEK-293 cells. We also have discovered that both
B-Raf and c-Raf-1 exist in HEK-293 cells, and we discuss below how
these Raf isozymes may participate in cross-talk between the PKA and
PKC pathways to influence ERK activity. These results provide a novel
mechanistic insight into pathways linking GPCRs to ERK activation. Our
data with endogenous purinergic receptors show both similarities and
differences (discussed below) with previous studies that utilized
transiently overexpressed receptors. A caveat to the use of
overexpression is the possibility that it may result in abnormal coupling.
One interesting observation in this study is the differential effects
of the two closely related bisindolylmaleimide PKC inhibitors, GF
109203X and Ro 31-8220. Whereas both inhibitors effectively blocked ERK
activation by phorbol 12-myristate 13-acetate, only Ro 31-8220 attenuated P2Y2- and A23187-mediated responses. Both compounds have been reported to be potent and selective PKC inhibitors. Nevertheless, it has been recently noted that these two PKC inhibitors have differential actions and distinct pharmacological properties (41-44). Ro 31-8220 is a much more potent inhibitor of PKC -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
and PKA on Raf isoforms.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-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
-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).
RESULTS
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Fig. 1.
Endogenous A2BAR-mediated cyclic
AMP accumulation in HEK-293 cells. A, dose-dependent
accumulation of cAMP in HEK-293 cells in response to the non-selective
AR agonist NECA. Cells were stimulated for 15 min with various
concentrations of NECA in the presence of 20 µM Ro
20-1724. Cyclic AMP levels were measured as described under
"Experimental Procedures". B, cyclic AMP accumulation in
response to various AR agonists and the effects of different AR
antagonists. All data points represent the mean ± S.E. of
triplicate determinations. The data shown are representative of three
independent experiments.
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Fig. 2.
Ca2+ mobilization in HEK-293
stimulated with AR agonists. Intracellular Ca2+
concentrations were measured in suspended cells loaded with FURA-2/AM.
A, NECA (1 µM)-stimulated Ca2+
mobilization in the absence or presence of 100 µM
enprofylline, 100 µM theophylline, or 1 µM
WRC0571. B, Ca2+-mobilization in response to
selective AR agonists, 1 µM CPA (A1AR),
IB-MECA (A3AR), or CGS21680 (A2AAR).
C, blockade of NECA or UTP-simulated Ca2+
mobilization by the phospholipase C inhibitor U73122. HEK-293 cells
loaded with Fura-2 were treated with either dimethyl sulfoxide
(vehicle) or U73122 (10 µM) for 15 min prior to the
stimulation with 10 µM NECA or 100 µM UTP.
The data shown are representative of at least three independent
experiments.
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Fig. 3.
Time course and dose dependence of
NECA-stimulated ERK1/2 activation in HEK-293 cells. A,
serum-starved HEK-293 cells were incubated at 37 °C with vehicle,
dimethyl sulfoxide (NS, not stimulated), or 10 µM NECA for the indicated times prior to determination of
ERK1/2 phosphorylation. B, serum-starved HEK-293 cells were
stimulated for 5 min with either vehicle or various concentrations of
NECA prior to determination of ERK1/2 phosphorylation. Data are
expressed as the fraction of maximal NECA-stimulated responses. Each
point is the mean ± S.E. of pooled data from at least three
independent experiments. The insets in A and
B are typical immunoblots representing phospho-ERK1/2
(top) and total ERK2 (bottom).
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Fig. 4.
NECA-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.
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Fig. 5.
NECA-stimulated ERK1/2 activation in HEK-293
cells is MEK1/2 and Ras dependent. A, blockade of
NECA-stimulated ERK1/2 activation by the MEK1/2 inhibitor PD 098059. Serum-starved HEK-293 cells were preincubated at 37 °C for 20 min
with 50 µM PD 098059 prior to stimulation with 10 µM NECA for 5 min. ERK1/2 phosphorylation
(top) and total ERK2 (bottom) was then determined
by immunoblotting. B, dominant negative Ras-N17 inhibits
NECA-stimulated MAPK activation. HEK-293 cells were transiently
co-transfected with plasmid DNA encoding FLAG-tagged ERK2 (2 µg/plate) plus either empty vector pcDNA3 (5 µg/plate, control)
or Ras-N17 (5 µg/plate). Serum-starved cells were then stimulated for
5 min with 10 µM NECA, 100 µM UTP, or 12.5 ng/ml EGF. FLAG-tagged ERK2 was immunoprecipited with anti-FLAG M2 gel
and phosphorylation of tagged ERK2 (top), the total
immunoprecipated tagged ERK2 (middle), and expression of
Ras-N17 (bottom) were determined by immunoblotting using
appropriate antibodies. The data shown are representative of three
independent experiments with similar results.
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Fig. 6.
Insensitivity of NECA-stimulated ERK1/2
activation to the tyrosine kinase inhibitor, genistein.
Serum-starved HEK-293 cells were pretreated with 100 µM
genistein at 37 °C for 20 min prior to stimulation with vehicle
(dimethyl sulfoxide), 10 µM NECA, 100 µM
UTP, or 25 ng/ml EGF. ERK1/2 phosphorylation was determined by Western
analysis. The data are normalized to the NECA-stimulated responses and
each bar is the mean ± S.E. of pooled data from three
independent experiments. Representative immunoblots of phospho-ERK2
(top) and total ERK2 (bottom) are shown.
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Fig. 7.
Effect of the protein kinase A (PKA)
inhibitor H-89 on NECA- or forskolin-stimulated ERK1/2 activation.
Serum-starved HEK-293 cells were preincubated at 37 °C for 30 min
with 10 µM H-89 prior to stimulation with 10 µM forskolin, 10 µM NECA, or 100 µM UTP and ERK1/2 phosphorylation was then determined by
Western analysis. The data are normalized to the NECA-stimulated
responses and each bar is the mean ± S.E. of pooled
data from multiple experiments. DMSO, dimethyl
sulfoxide.
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Fig. 8.
NECA-stimulated ERK1/2 activation in HEK-293
cells is sensitive to the PLC inhibitor U73122. Serum-starved
HEK-293 cells were preincubated at 37 °C for 15 min with either
vehicle (dimethyl sulfoxide) or 10 µM U73122. Cells were
then stimulated with vehicle (dimethyl sulfoxide, DMSO,
control), 10 µM NECA, or 100 µM UTP, and
ERK1/2 phosphorylation was determined by Western analysis. The data are
normalized to the NECA-stimulated responses and each bar is
the mean ± S.E. of pooled data from three independent
experiments. Representative immunoblots of phospho-ERK2
(top) and total ERK2 (bottom) are shown.
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Fig. 9.
NECA-stimulated ERK1/2 activation is not
sensitive to PKC inhibitors GFX or Ro 31-8220. Serum-starved
HEK-293 cells were preincubated at 37 °C for 15 min with dimethyl
sulfoxide (DMSO), PKC inhibitors GF 109203X (2 µM, A), or Ro 31-8220 (10 µM,
B and C) prior to stimulation with the
appropriate agonists for 5 min (NECA (10 µM), UTP (100 µM), A23187 10 (µM), forskolin (10 µM), phorbol 12-myristate 13-acetate (100 nM), or UTP (100 µM) + forskolin (10 µM)). ERK1/2 phosphorylation was determined by Western
analysis. The data are normalized to the NECA (A and
B) or UTP (C)-stimulated responses and each
bar is the mean ± S.E. of pooled data from three
independent experiments. Representative immunoblots of phospho-ERK2
(top) and total ERK2 (bottom) are shown.
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Fig. 10.
A2BAR-initiated sustained ERK1/2
activation in canine BR mast cells. A, serum-starved
canine BR mast cells were stimulated for various times as indicated
with 10 µM NECA. ERK1/2 phosphorylation (top)
and total ERK2 (top) were determined by immunoblotting.
B, comparison of ERK1/2 activation by NECA with other AR
selective agonist and blockade of NECA-induced ERK activation by
enprofylline. Serum-starved canine BR mast cells were stimulated for 10 min with indicated stimulants ± 100 µM
enprofylline. ERK1/2 phosphorylation (top) and total ERK2
(top) were determined by immunoblotting.
DISCUSSION
(106-169 nM versus 5800 nM) than is
GF 109203X (45-48), whereas they are almost equipotent as inhibitors
of other PKC isoforms. This raises the interesting possibility that
Gq/11-coupled receptors may selectively activate PKC
to
regulate ERK1/2 activity in HEK-293 cells. The presence of PKC
in
HEK-293 cell membranes has recently been demonstrated by Western
blotting (49). Of note, in this regard, is the finding that in rat
astrocytes, ERK activation by endogenous P2Y receptors, was also
inhibited by Ro 31-8220, but not by Gö 6976, an inhibitor of PKC
and
1 isozymes which does not affect PKC
,
, or
(50).
It has been reported that in vascular smooth muscle cells, PKC
,
mediates Ras-dependent ERK1/2 activation induced by Ang-II
(51). Involvement of Ras in PKC
activation both in vivo
and in vitro has also been reported (52). In a previous
study (23), it has been concluded that GPCRs in HEK-293 cells do not
activate ERK via PKC because these responses are insensitive to GF
109203X. Based on our data, we propose that PKC
is the primary PKC
isozyme that contributes to ERK stimulation due to activation of
A2BAR and P2Y2 receptors in HEK-293 cells (Fig.
11).
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Fig. 11.
Model for the regulation of ERK1/2 activity
by A2BAR and P2Y2 receptors in HEK-293
cells. Both A2BARs and P2Y2 receptors
activate ERK by a pathway that is thought to include Gq/11,
a genistein-insensitive tyrosine kinase, Ras, B-Raf, c-Raf-1, and MEK.
Forskolin and the A2BAR elevate cyclic AMP and can activate
ERK via a pathway that is attenuated by PKC ( ). ERK activation by
endogenous purinergic receptors is stimulated in part by PKC (
). The
model accounts for the effects of NECA, UTP, forskolin, H-89 and Ro
31-8220 (see text).
Another interesting aspect of this study is evidence of cross-talk
between PKA and PKC in modulating ERK activation by
A2BARs. We show that ERK activation by the P2Y2
receptor, but not by A2BAR, is inhibited by Ro 31-8220. The
major difference between P2Y2 receptors and
A2BARs is that only A2BARs couple via
Gs to activate adenylyl cyclase. This suggests that a
common Gq/11 and PKC
pathway utilized by
A2BARs and P2Y2 receptors may be modulated by
cyclic AMP. We show that cyclic AMP has several effects on ERK
signaling in HEK-293 cells (Fig. 11). ERK is activated by forskolin and
this response is abolished by the PKA inhibitor, H-89, and enhanced by
the PKC inhibitor Ro 31-8220. The addition of forskolin converts
UTP-induced activation of ERK from being attenuated, to being
unaffected by Ro 31-8220. According to our model (Fig. 11) Ro 31-8220 can inhibit PKC
-mediated activation of ERK, but enhance a cyclic
AMP-dependent B-Raf-induced activation. Hence, when cyclic
AMP is elevated by NECA or UTP plus forskolin, Ro 31-8220 has little
net effect on ERK activation.
According to this scenario, one would expect that cyclic AMP should
contribute to ERK activation mediated by A2BARs. However, the PKA inhibitor, H-89, although inhibitory to forskolin-induced activation of ERK, only marginally affects NECA-induced responses. To
account for this, our model draws on previous studies which show cyclic
AMP can have either an inhibitory (e.g. in Rat-1 or NIH3T3
cells) or stimulatory effect (in PC12 cells) on ERK activation depending on the cell type involved. Work by Vossler et al.
(33) suggested that cyclic AMP decreases ERK stimulation by inhibiting c-Raf-1 activation, whereas it increases ERK activity by activating B-Raf. Consistent with this notion, we have detected by Western blotting both B-Raf and c-Raf-1 in the HEK-293 cells used in this study. Our demonstration that forskolin can activate ERK in HEK-293 cells, likely via B-Raf, is confirmatory of previous work by Daaka et al. (53). The lack of an effect of elevated cAMP on
A2BAR-mediated ERK activation might result from a balance
between opposing effects of cyclic AMP on ERK activation,
i.e. stimulation via B-Raf activation and inhibition via
c-Raf-1 inactivation. We note that the magnitude of cyclic AMP
accumulation induced by NECA (10 µM) is lower than that
induced by forskolin (10 µM). This may also contribute to the small apparent contribution of cAMP to A2BAR-mediated
ERK activation, along with the fact that PKC activation by NECA counteracts the effect of PKA on B-Raf.
In the present study, we show that ERK activation by both
A2BARs and P2Y2 receptors requires functional
Ras. The mechanisms of Ras activation by various GPCRs remain poorly
characterized, particularly for Gq-coupled receptors. By
overexpressing the carboxyl terminus of the -adrenergic receptor
kinases 1 (
ARKct), a scavenger of G
released from activated G
proteins, it has been shown that Ras activation by
Gi-coupled receptors is mediated by G
(54). However,
in the case of Gq-coupled receptors, the involvement of
G
is controversial (54, 55). We found that transfection of
HEK-293 cells with
ARKct has little effect on co-transfected epitope-tagged ERK activation by A2BARs and
P2Y2 receptors in HEK-293 cells (data not shown). This
suggests that G
, but not G
, is principally responsible for
Ras-dependent MAPK activation following the stimulation of
purinergic receptors. It is possible that Gi-coupled
receptors release more and/or different
subunits than
Gq or Gs.
We also demonstrate herein that Ras-dependent ERK1/2
activation by either A2BARs or UTP receptors in HEK-293
cells does not require genistein-sensitive tyrosine kinases. Although
this is somewhat unexpected, it is not without precedent.
Gi-coupled m2 muscarinic receptors expressed in Rat-1
fibroblasts activate ERK in a Ras- and Raf-dependent
manner, and this response is insensitive to inhibition by genistein
(56). The same holds true for Gi-coupled 5-HT1A
receptors expressed in Chinese hamster ovary cells (22). In contrast,
ERK activation by transiently transfected Gi-coupled m2
muscarinic receptors in COS cells, Gi-coupled
lysophosphatidic acid receptors in Rat-1 fibroblast (57), or
Gq/11-coupled 1B-adrenergic receptors in
HEK-293 cells (23) require both Ras and genistein-sensitive tyrosine
kinases. It is possible that overexpression of these receptors
influences coupling to ERK. Although this discrepancy cannot be
definitively explained at present, these findings support the current
view that GPCRs modulate ERK activity in a cell-type, receptor-specific, and possibly a receptor-density dependent manner.
The issue of how ERK signaling specificity is achieved, especially in the case of multiple GPCRs apparently coupled to the same type of G-protein in the same cell type, is addressed in a recent study by Mitchell et al. (58) describing the activation of phospholipase D by various Gq/11-coupled receptors. These investigators identified a specific structural motif (NPXXY versus DPXXY) in a subset of Gq/11-coupled receptors, which is important for Rho-mediated phospholipase D activation. A similar scenario is conceivable for ERK activation by GPCRs coupled to the same G proteins. G protein-coupled receptors may couple to additional cellular constituents other than G proteins to transduce signals and influence ERK activation in a receptor-specific way. Daaka and co-workers (59, 60) recently presented evidence to address the importance of GPCR endocytosis in the activation of ERKs. In view of the various pathways used for GPCR internalization, it is possible that specific mechanisms of ERK activation by various GPCRs might also reflect differences in receptor endocytosis.
In summary, all of our data fit the scheme depicted in Fig. 11.
P2Y2 receptors couple via Gq/11 only, and
activate ERK via pathways including genistein-insensitive tyrosine
kinases, phospholipase C, PKC , Ras, and Raf. In contrast,
A2BARs couple to both Gs and Gq/11,
and the Gs signaling branch exerts both stimulatory and
inhibitory effects on Gq/11-mediated ERK activation via
cyclic AMP-dependent PKA. Gq/11-mediated PKC
activation has inhibitory effects on cAMP-dependent ERK
activation. Hence, there exists an interesting cross-talk between PKC
and PKA signaling pathways in regulating ERK activity by
Gs- and Gq/11-coupled receptors in HEK-293
cells. It is notable that extracellular ATP released from cells is
rapidly broken down to adenosine by ectonucleotidases. Since both P1
and P2 receptors are simultaneously activated, cross-talk between the
ERK activation pathways mediated by different purinergic receptors
assumes an even broader role in a physiological context.
In the present study, we also demonstrate that A2BARs
trigger sustained ERK activation in canine BR mast cells. Although this response is initiated by A2B adenosine receptors, the
sustained phase of activation may not be solely maintained by
A2BARs. The mediators released in response to
A2BAR activation of mast cells may play a role in promoting
sustained ERK activation. The cellular implications of transient
versus sustained ERK activation are different. In PC12
neuronal cells, sustained ERK activation by NGF leads to
differentiation and is associated with ERK translocation from the
cytosol to the nucleus, whereas EGF-mediated transient ERK activation
leads to cell proliferation but not differentiation or ERK nuclear
translocation (16). What might be the function of
A2BAR-mediated ERK activation in mast cells? It was
reported that in the rat mast cell line (RBL-2H3), activation of IgE
receptors triggers ERK activation, and this signaling is responsible
for the release of arachidonic acid and the regulation of cytokine gene
expression but not the release of secretory granules (which contains
histamine, ATP, etc.) (11, 12). Activation of PKC and an increase in
intracellular Ca2+ provide sufficient signals for mast cell
degranulation (61). Based on these observations, we hypothesize that
ERK activation may be responsible for AR-mediated release of
arachidonic acid and promotion of cytokine production (such as
A2BAR-mediated interleukin-8 synthesis in human mast cell
line HMC-1 (6)). The sustained phase ERK activation may also be
important for AR regulation of mast cell proliferation and differentiation.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Walter J. Koch (Duke University)
for kindly providing mini-gene construct encoding ARKct and Drs.
Barbara Hettinger and Michael Broad (University of Virginia) for
insightful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants RO1-HL37942 (to J. L.) and GM 47332 (to M. W.).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.
To whom correspondence should be addressed: Box MR4, 6012 Health Sciences Center, University of Virginia, Charlottesville, VA
22908. Tel.: 804-924-5600; Fax: 804-982-3162; E-mail:
jlinden{at}virginia.edu.
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ABBREVIATIONS |
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The abbreviations used are:
AR, adenosine
receptor;
CPA, N6-cyclopentyladenosine;
CGS21680, 2-p-(2-carboxyethyl)phenethylamino-5'-ethylcarboxaminoadenosine;
IB-MECA, N6-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine;
NECA, 5'-N-ethylcarboxamidoadenosine;
theophylline, 1,3-dimethylxanthine;
enprofylline, 3-propylxanthine;
WRC0571, C8-(N-methylisopropyl)-amino-N6-(5'-endohydroxy)-endonorbornan-2-yl-9-methyladenine;
PKA, protein kinase A;
EGF, epidermal growth factor;
ERK, extracellular
signal-regulated kinase;
PKC, protein kinase C;
Ro 20-1724, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone;
PD098059, 2'-amino-3'-methoxyflavone;
U73122, 1-{6-[(17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino]hexyl}-1H-pyrrole-2,5-dione;
GF 109203X (GFX), bisindolylmaleimide I;
Ro 31-8220, 3,1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide
methane sulfonate;
MAPK, mitogen-activated protein kinase;
GPCR, G
protein-coupled receptor.
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REFERENCES |
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