 |
INTRODUCTION |
Extracellular nucleotides can interact with cell surface P2
receptors both in the central nervous system and in peripheral tissues
to produce a broad range of physiological effects. The P2 family is
divided into two main types as follows: the P2X receptors are
ligand-gated ion channels, and the P2Y receptors are G protein-coupled (1, 2). Part of the present study describes the signaling pathways of
the P2Y1 receptor, the first member of the P2Y family to be
identified (3). The P2Y1 receptor is widely distributed and
has been described in mammalian heart, vascular, liver, kidney, prostate, gastrointestinal, pulmonary, connective, and immune tissues
(4, 5). It has also been identified in skeletal muscle and appears to
be the most abundant P2 receptor in the nervous system (5-7). ADP and
more potently 2-methylthio-ADP (2-MeSADP)1 are agonists at
the P2Y1 receptor, but its activation by ATP and 2-MeSATP
has been controversial. Due to the ready conversion of triphosphates by
ectonucleotidases to the aforementioned agonistic diphosphates, some
earlier findings are in doubt. Thus, it is important to maintain
totally the triphosphate integrity by a constant regenerating system
(8, 9), which has been included in the experimental design of the
present study. With such precautions in place, the recombinant
P2Y1 receptor can be activated by ATP and 2-MeSATP (10,
11), whereas others (9) have found these ligands to be antagonists in
other cell systems. This difference has been proposed to depend
critically on the degree of P2Y1 receptor reserve (10,
11).
The second messengers generated by the P2Y1 receptor are
due to the activation of phospholipase C
leading to the formation of
diacylglycerol as well as inositol trisphosphate (12, 13) and
mobilization of intracellular Ca2+ (9, 10), suggesting that
the subsequent activation of protein kinase C (PKC) is likely. These
responses are insensitive to pertussis toxin, and the G protein
involved has been identified as G11 in the case of the
turkey erythrocyte (14), but Gq can also act at the
P2Y1 receptor in some systems including the human platelet (15). Furthermore, a primary signaling action of the P2Y1
receptor in neurons is the closing of an N-type Ca2+
channel (11), and in platelets there is some suggestion that the
P2Y1 receptor is coupled to the RhoA-ROCK pathway (16). It
is known that activation of PKC isoforms by G protein-coupled receptors
can stimulate the extracellular signal-regulated kinases (ERKs), which
are members of the mitogen-activated protein (MAP) kinase family (17).
For the P2Y receptors, activation of the ERK cascade has been shown in
several cell types including astrocytes (18, 19), endothelial cells
(20, 21), vascular smooth muscle cells (22), and renal mesangial cells
(23). However, since the cells studied co-express several types of P2Y
receptors or have an unknown complement of purinoceptors, the
downstream events observed have not identified those associated with a
single, molecularly defined, P2Y receptor.
The MAP kinases are proline-directed serine/threonine kinases that have
been classified into at least four subfamilies as follows: ERKs,
stress-activated protein kinases (SAPKs), p38 kinases, and BMK1/ERK5
(17). Whereas ERKs are implicated in cell growth as well as
differentiation, SAPKs and p38 appear to play a role in regulating the
cell death machinery (24). Whereas the pathway linking cell surface
receptors to ERKs has been partially elucidated, the mechanism of
activation of p38 and SAPKs is poorly understood. This is particularly
so for members of the G protein-coupled receptor family, which have
only recently been shown to utilize these alternative MAP kinase
cascades for transduction purposes. Activation of p38 and SAPKs has
been demonstrated following stimulation of the
Gq/G11-coupled m1 and
Gi-coupled m2 muscarinic acetylcholine
receptors (25, 26). More recent studies have also shown p38 activation
in rat glomerular mesangial cells following stimulation with UTP and ATP, possibly mediated through the P2Y2 receptor (27). In
addition, stimulation of P2Y2 receptors in C6 glioma cells
(28) and/or P2Y4 receptors in rat glomerular mesangial
cells can induce proliferation (29). At present apoptosis initiated by
nucleotides is known only for a P2X receptor, being a prominent
consequence of P2X7 receptor activation in human
macrophages and leukocytes as well as in mesangial, dendritic, and
microglial cells (30, 31). As with most examples of apoptosis, the
P2X7 receptor-initiated cascade, which includes the
coupling of the ion channel to the SAPK-signaling cascade (32),
involves a defined sequence of phenotypic changes that culminate in
death only several hours after the exposure to ATP.
The goal of this study was to examine the ability of the human
recombinant P2Y1 receptor, heterologously expressed in
human astrocytoma cells (1321N1, containing no endogenous P2Y
receptors) to stimulate the MAP kinase transduction cascades. A
specific antagonist of the P2Y1 receptor was applied to
confirm the authenticity of the responses observed. In addition, the
time course of the MAP kinase activity was also determined. There is
much evidence to suggest that the duration of ERK activity is
critically important for determining functional outcome (33, 34), and
in every case examined thus far, only sustained ERK activation induces cytoplasmic nuclear migration (35, 36). Prolonged stimulation of ERK
will therefore have very different consequences for gene expression
compared with that of transient activation. Part of this study was thus
to determine if the duration of the other MAP kinase cascades is
similarly important for controlling transcriptional events. In
addition, it was determined whether the P2Y1
receptor-mediated MAP kinase activities could be correlated with either
a proliferative or an apoptotic functional response.
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EXPERIMENTAL PROCEDURES |
Materials--
The 1321N1 astrocytoma cell line heterologously
expressing the human P2Y1 receptor (5) was a generous gift
from Dr. S. P. Kunapuli (Temple University, Philadelphia). All
tissue culturing media and reagents were purchased from Life
Technologies, Inc., and plasticware was from Costar. Carbamylcholine
chloride (carbachol), creatine phosphokinase, creatine phosphate,
2-MeSADP, 2-MeSATP, and adenosine-2'-phosphate-5'-phosphate (A2P5P)
were purchased from Sigma. ADP and hexokinase were from Roche Molecular
Biochemicals. Antibodies specific to ERK1 and ERK2 were obtained from
Santa Cruz Biotechnology. Polyclonal antibodies specific for the dually phosphorylated and hence active forms of ERK1 and ERK2 (at
Thr202 and Tyr204),
,
, and
isoforms
of p38 (at Thr180 and Tyr182), and SAPK family
members (at Thr183 and Tyr185), together with
antibodies to p38 and SAPKs with a specificity for the kinases
independent of their phosphorylation state, were all obtained from New
England Biolabs. Antibodies to the transcription factors c-Jun, Elk-1,
and ATF-2 as well as those to the phosphorylated forms of these
proteins were also supplied by New England Biolabs. The PKC inhibitors,
Gö 6976 and Ro 32-1432, as well as Bordetella pertussis toxin, the MEK1 inhibitor, PD 98059, the Src inhibitor, PP1, and the phosphatidylinositol 3-kinase (PI 3-K) inhibitor, LY
294002, were all from Calbiochem. Antibodies to the G protein
subunits were from Santa Cruz Biotechnology. An anti-MKK4 (SEK1) antibody was obtained from New England Biolabs. Antibodies specific for
Ras (L2 region) and human Ha-RAS cDNA (dominant
negative S17N mutant) in pUSEamp, together with the empty vector, were
supplied by Upstate Biotechnology, Inc. Staurosporin, FITC-conjugated
annexin V, and a caspase-3 substrate that is cleaved to release a
colorimetric product were all from CLONTECH.
Enzymatic Conversion of Tri- or Diphosphate
Contamination--
ATP and ADP analogues are metabolically unstable
and can be degraded by various ectonucleotidases present on cells. In
addition, commercially available nucleotides often contain other
nucleotides as by-products. Therefore, enzymatic systems that
regenerate degraded nucleotides were routinely used in all experimental
procedures carried out in this study. To eliminate the diphosphate
contamination of 2-MeSATP, the creatine phosphokinase-regenerating
system was used according to a method described previously (9). Thus, 1 mM stock solutions of 2-MeSATP were treated with 20 units
ml
1 creatine phosphokinase in the presence of
10 mM creatine phosphate for 90 min at 37 °C. In order
to ensure the purity of ADP and 2-MeSADP, 1 mM stock
solutions were treated with 10 units ml
1
hexokinase, 0.1 M glucose for 60 min at room temperature to
convert the triphosphate contamination to diphosphates (37). The
appropriate enzymes (with their substrates) were also included (at 1 unit ml
1) in the culture medium during all experiments.
Tissue Culture--
Human astrocytoma cells (1321N1) stably
expressing the human recombinant P2Y1 receptor were
cultured in Dulbecco's modified Eagle's medium/Nutrient Mix F-12
medium (1:1) containing Glutamax I, pyridoxine hydrochloride, 10%
fetal bovine serum, and 600 µg ml
1
geneticin sulfate (G-418). The cells were maintained at 37 °C in a
humidified atmosphere (95% air, 5% CO2) and passaged by
trypsinization every 4-5 days. For MAP kinase activity experiments,
cells were plated in 6-well plates at an original seeding density of
200,000 cells per well and cultured to 80% confluence.
Activation of MAP Kinases by Nucleotides--
Cells were
serum-starved for 4 h before incubation with incomplete media
containing various drug treatments for the appropriate times at
37 °C. Reactions were terminated by removing the media and adding
150 µl of 3× concentrated Laemmli sample buffer. Following solubilization, the well contents were transferred to Eppendorf vials,
and the wells were rinsed with 100 µl of distilled H2O. Equivalent amounts of protein were electrophoretically resolved on 10%
polyacrylamide gels. Following electrophoretic transfer onto
nitrocellulose (0.22 µm) using a semi-dry blotter, the membrane was
washed briefly in Tris-buffered saline (TBS) and saturated overnight in
TBS supplemented with 0.1% Tween 20 and 5% dried milk. For detection
of the phosphorylated forms of the kinases, the nitrocellulose membrane
was incubated with a 1:800 dilution of the anti-phosphospecific
antibodies. Primary incubations were for 1 h at 22 °C in TBS
containing 0.1% Tween 20 (TBST) followed by washing five times for 10 min each in TBST. Membranes were incubated for 1 h at 22 °C
with a 1:3,000 dilution of the appropriate horseradish
peroxidase-conjugated secondary antibody in TBST containing 5% dried
milk. Excess antibody was removed by washing as above, and
immunocomplexes were visualized using enhanced chemiluminescence detection, according to the manufacturer's instructions (Amersham Pharmacia Biotech). In order to substantiate the consistency of protein
content between treatment groups, the membranes were re-probed with
phosphorylation state independent antibodies to ERK1 and ERK2 (1:1,000
dilution) or SAPK/p38 (1:500 dilution) for 1 h at 22 °C and
processed as above. The Western blots shown are representative of three
separate experiments, and each panel is taken from a single immunoblot.
Phosphorylation of Transcription Factors--
Samples obtained
from the 2-MeSADP time course experiments were separated on 10%
polyacrylamide gels and transferred onto nitrocellulose membrane as
described above. The membrane was incubated with primary antibodies
specific to the phosphorylated forms of the transcription factors
ATF-2, Elk-1, and c-Jun (1:400 dilution) for 1 h at 22 °C. The
transcriptional activity of c-Jun is regulated by phosphorylation at
Ser63 and Ser73 by SAPKs. Antibodies specific
to both these phosphorylation sites were used. Elk-1 is phosphorylated
by ERK1 and ERK2 at a cluster of Ser/Thr motifs at its COOH terminus,
and phosphorylation of Ser383 (to which the antibody was
raised) has been shown to be critical for transcriptional activation.
Activation of ATF-2 requires phosphorylation of Thr69 and
Thr71, and these sites are both substrates for the p38
kinase as well as for the SAPKs. The antibody used was raised to a
synthetic phospho-Thr71 peptide. For detection of total
protein, phospho-independent antibodies to the transcription factors
were used at a 1:500 dilution. The Western blots shown are
representative of three separate experiments, and each panel is taken
from a single immunoblot.
Detection of the P2Y1 Receptor and G Protein
Complement--
Whole cell extracts from 1321N1 human astrocytoma
cells expressing the human P2Y1 receptor were separated on
10% polyacrylamide gels and transferred onto nitrocellulose. Membranes
were incubated with serial dilutions of an antibody that specifically
recognizes the COOH-terminal of the human P2Y1 receptor
(peptide sequence CTLNILPEFKQNGDTSL) or with antibodies recognizing
different G protein
subunits (1:250 dilution). All primary
incubations were for 1 h at 22 °C and immunoreactivity was
detected as described above.
Immunocytochemistry--
Astrocytoma cells (1321N1) stably
expressing the recombinant P2Y1 receptor were grown on
poly-L-lysine (100 µM
ml
1)-treated glass coverslips in 12-well
plates until they reached ~70% confluence. The media were removed
from the wells, and the coverslips were rinsed for 5 min (times three)
with phosphate-buffered saline (PBS), fixed with 2% formaldehyde in
PBS for 30 min, and washed as above. The cells were incubated for 30 min in 500 µl of blocking solution (PBS containing 3% goat serum,
1% bovine serum albumin, and 0.1% Triton X-100) followed by overnight
incubation at 4 °C with the primary antibody recognizing the
P2Y1 receptor (1:200 dilution in blocking solution).
Control coverslips were incubated with blocking solution alone. After
three washes for 5 min each in PBS, the cells were incubated with 500 µl of the secondary antibody conjugated to cyanine 3 (at 1:1,000
dilution, Sigma) in blocking solution for 1 h at 22 °C. After
three washes for 5 min each in PBS, the coverslips were removed from
the wells, dipped in distilled water, and dried before being mounted
onto slides using an antifade agent (DAKO). The fluorescence was
visualized using a Nikon Optiphot-2 microscope.
Expression Plasmids--
Dominant negative human MKK4 (K95R) was
constructed as described previously (38), and the full-length cDNA
was cloned into the mammalian expression vector, pCMV. Human
Ha-RAS (S17N) cDNA was inserted as an EcoRI
fragment into pUSEamp also under the control of the cytomegalovirus
promoter. Transfections were performed using the
LipofectAMINETM reagent according to the protocol suggested
by the manufacturer (Life Technologies, Inc.). Briefly, astrocytoma
cells (1321N1) stably expressing the recombinant P2Y1
receptor at 50% confluence were transfected in serum-free media with 2 µg of DNA following complex formation with
LipofectAMINETM reagent. The DNA-containing media were
removed following incubation for 3 h at 37 °C, and the cells
were incubated with complete medium. Gene expression using immunoblot
analysis as described above was determined immediately prior to drug
additions, ~48 h post-transfection using a primary antibody
concentration of 1:1,000.
Annexin V Binding--
In normal, non-apoptotic cells,
phosphatidylserine is segregated to the inner leaflet of the plasma
membrane. During early stages of apoptosis, this asymmetry collapses,
and phosphatidylserine becomes exposed on the outer surface of cells
(39). Annexin V is a protein that preferentially binds to
phosphatidylserine in a Ca2+-dependent manner.
Binding of annexin V in conjunction with propidium iodide exclusion to
establish membrane integrity was used to identify apoptotic cells.
Astrocytoma cells heterologously expressing the recombinant
P2Y1 receptor were grown on poly-L-lysine (100 µM ml
1)-treated glass
coverslips in 12-well plates until ~40% confluence was reached. The
cells were serum-starved for 1 h before a 5-h incubation in the
presence of various agents. The media were removed from the wells, and
the coverslips were rinsed in ice-cold PBS. The coverslips were
incubated for 15 min in the dark at 22 °C with 200 µl of binding
buffer (150 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 1.8 mM CaCl2, 10 mM NaHEPES, pH 7.4) containing 0.5 µg
ml
1 annexin V-FITC and 10 µg
ml
1 propidium iodide. The coverslips were
washed twice in binding buffer, dried, and mounted onto microscope
slides as described above. Annexin V-FITC binding and propidium iodide
incorporation were detected using either a Nikon Optiphot-2 microscope
or a confocal laser scanning microscope (Zeiss LSM 510) with FITC
excitation at 488 nm, emission 505-550 nm, and propidium iodide
excitation at 543 nm, emission >560 nm. Maximum projection images of z
series are shown.
Determination of Caspase-3 Activation--
Cytosolic
aspartate-specific proteases, called caspases, are responsible for
the deliberate disassembly of a cell into apoptotic bodies. Caspases
are present as inactive pro-enzymes, most of which are activated by
proteolytic cleavage. Both caspase-8 and caspase-9 can activate
caspase-3 by proteolytic cleavage, which can in turn cleave vital
cellular proteins leading to apoptosis (40). In this study, we have
determined caspase-3 activity as an indicator of apoptotic induction.
After challenge with appropriate agonists, 1 × 106
cells (including those that had dislodged from the adherent monolayer) were harvested by centrifugation for 30 s at 100 × g, and the pellets were resuspended in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM
dithiothreitol, 5 mM EDTA, 5 mM EGTA, 1%
Triton X-100, pH 7.5), incubated for 15 min at 37 °C, and
centrifuged at 12,000 × g for 20 min at 4 °C. To
assess supernatant caspase-3-like activity, 0.3 ml of lysate was
combined with buffer (100 mM NaHEPES, 10% glycerol, 1 mM EDTA, 5 mM dithiothreitol, pH 7.5) and
colorimetric caspase-3 substrate (IETD-pNA) to a final concentration of
20 µM. Samples were incubated in the dark at 37 °C,
and absorbance was measured at 400 nm at 10-min intervals using a
PerkinElmer Life Sciences spectrophotometer. Data were plotted and
slopes calculated along the linear portion of the curve (at least three separate measurements). Data are presented as slope in arbitrary units
and are expressed as the arithmetic means ± S.E. of the mean
(n = 3). Statistical analysis was by Student's
t test.
Determination of Cell Numbers--
Cells were harvested
following incubation for the appropriate period by washing the
monolayers in PBS and adding 0.05% trypsin, 0.02% EDTA solution for
2-5 min, and the single cell suspension counted using a Coulter
CounterTM model Z1. Results are expressed as the mean cell
number (± S.E.) harvested from a single well (n = 3, 3 replicates per test group). Statistical analysis was by Student's
t test.
 |
RESULTS |
Detection of the Human Recombinant P2Y1 Receptor and
the G Protein
Subunit Complement of Astrocytoma (1321N1)
Cells--
The heterologous expression of the human recombinant
P2Y1 receptor in astrocytoma cells (1321N1) was confirmed
by immunocytochemistry as well as by Western analysis using an
anti-peptide antibody directed against the COOH-terminal tail of the
human P2Y1 receptor. This antibody is selective for the
P2Y1 receptor subtype and recognizes both the human and rat
orthologues. Fixed cells incubated without the primary antibody showed
no detectable staining, whereas those incubated with the
anti-P2Y1 receptor antibody showed marked fluorescence that
was localized to the membrane surface (Fig.
1A). Whole cell protein
extracts prepared from the selected clonal line showed concentration-dependent immunoreactivity following Western
analysis with the P2Y1 receptor-specific antibody (Fig.
1B). The electrophoretic mobility of the broad band detected
had an apparent molecular mass of 45-55 kDa. No detectable staining
was observed following Western analysis of protein from wild-type
1321N1 astrocytoma cells (data not shown). Whole cell extracts from
astrocytomas expressing human P2Y1 receptors were also
analyzed using antibodies specific for some relevant subtypes of the
subunits of G proteins (Fig. 1C). Immunoreactive bands
of the predicted molecular masses were identified for
G
13, G
o, and the short form of
G
s. A double band was detected using an antibody that
cross-reacts with both G
q and G
11. No
detectable immunoreactivity was observed with an antibody specific for
G
i2, but a strong band was observed following analysis
with an antibody that recognizes all three forms of
G
i.

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Fig. 1.
Detection of the heterologously expressed
human recombinant P2Y1 receptor and the complement of G
protein subunits in 1321N1 astrocytoma
cells. A, immunocytochemical localization of the human
P2Y1 receptor expressed in astrocytoma cells was determined
using the COOH-terminally directed anti-P2Y1 receptor
antibody (1:200 dilution) in the presence of Triton X-100. Control
cells were incubated without primary antibody. Scale bar, 50 µm. B and C, whole cell protein extracts
prepared from 1321N1 cells were electrophoretically separated on 10%
polyacrylamide gels. Following transfer onto nitrocellulose, the
membranes were probed either with serial dilutions of the
anti-P2Y1 receptor antibody (B) or with
antibodies directed against the subunits of G proteins (at 1:250
dilution except for s1 that was at a 1:100 dilution)
(C). The electrophoretic mobilities of marker proteins are
also shown (arrows). The data are representative of at least
three separate experiments.
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P2Y1 Receptor-mediated Effects on the Phosphorylation
Status of MAP Kinases--
To determine changes in the phosphorylation
status of the different MAP kinases upon activation of the human
P2Y1 receptor, whole cell protein extracts were analyzed by
Western blotting using antibodies specific for the dually
phosphorylated kinases and hence active forms. In serum-starved
astrocytoma cells expressing the P2Y1 receptor, the
immunoreactivity detected with antibodies selective for ERK1, ERK2,
p38, and the SAPK isoforms, independent of their phosphorylation
status, showed the expression of these proteins to be unaffected over
the time course studied (up to 4 h) and by the application of the
potent P2Y1 receptor agonist, 2-MeSADP (300 nM)
(Fig. 2). A single species could be
detected with the antibody to p38, and ERK1 and ERK2 had the predicted molecular mass of 44 and 42 kDa, respectively. However, several distinct entities could be observed following detection with the anti-SAPK antibody. The p54 and p46 members of this kinase family were
apparent. Other immunoreactive bands were also detected with mobilities
corresponding to 45 and 48 kDa (Fig. 2B).

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Fig. 2.
Effect of 2-MeSADP on the phosphorylation
status of ERK1 and ERK2 (A) or p38 and SAPKs
(B) in astrocytoma cells heterologously expressing
P2Y1 receptors. Whole cell extracts were prepared from
cells that had been serum-starved for 4 h
(T0) before incubation with incomplete media
(Basal) or 2-MeSADP (300 nM) for the times shown
in minutes. Samples were analyzed by Western detection following
separation on 10% polyacrylamide gels. Consistency of protein loading
was substantiated by determining the immunoreactivity of samples with
phosphorylation state independent antibodies (top panels).
Phosphorylation changes were demonstrated by detection with an antibody
to ERK1 and ERK2 that recognizes only the dually phosphorylated (at
Thr202 and Tyr204) and hence active forms
(ERK-P). Similarly, SAPK and p38 activations were assessed
using antibodies specific for the doubly phosphorylated forms of all
isoforms at residues Thr183 and Tyr185
(p46-P and p54-P shown) or Thr180 and
Tyr182 (p38-P), respectively.
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Under basal conditions over the time course studied, a slight
phosphorylation of ERK1 and ERK2 was detected that peaked 10 min
following application of the vehicle control and which had declined to
undetectable levels by 1 h (Fig. 2A). Application of
2-MeSADP (300 nM) induced a marked and rapid
phosphorylation of both ERK kinases that was detectable after 5 min and
had reached a maximal response at 15 min. Although the level of
phosphorylation subsequently declined, that detected 4 h following
drug application remained elevated over basal (Fig. 2A). To
substantiate the consistency of protein content, the immunoreactivity
of samples was also determined using phosphorylation state-independent
anti-ERK antibodies (Fig. 2A).
The phosphorylation of p38 in serum-starved 1321N1 cells was only
just detectable using the phosphospecific antibody, and the level of
this activity remained unchanged throughout the time course
investigated following application of either vehicle control (basal) or
2-MeSADP (300 nM) (Fig. 2B). Differential levels
of phosphorylation were detected for the SAPKs after serum starvation and that observed for the isoform with apparent molecular mass of 45 kDa was much more pronounced than that for the other SAPK isoforms
present (46, 48, and 54 kDa). Following application of vehicle alone,
none of the isoforms showed any change in their activity status over
the 4-h period investigated (Fig. 2B). A transient increase
in the phosphorylation of the 45-, 46-, and 54-kDa forms was observed
upon application of 2-MeSADP (300 nM), reaching a maximum
by 15 min and returning to basal levels by 1 h (Fig.
2B). In contrast, the activity status of the 48-kDa isoform
remained unaffected by 2-MeSADP (300 nM) over the time course studied. The level of immunoreactivity using phospho-independent antibodies was comparable for p38 and the p45 and p54 forms of the
SAPKs. The level detected for the remaining SAPK isoforms, however, was
much lower, particularly that with a molecular mass of 46 kDa. The
levels of the p38 and the SAPK proteins were essentially constant
across the time course studied and between basal and 2-MeSADP-treated
groups (Fig. 2B).
Effects of an Adenosine Triphosphate and ADP--
Activation of
the human recombinant P2Y1 receptor by 2-MeSATP or by ADP
(0.1-10 µM) induced concentration-dependent
phosphorylation changes in ERK1 and ERK2 (Fig.
3A). However, in contrast to
that evoked by 2-MeSADP (300 nM), the enhanced
immunoreactivity was transient in duration, with increased
phosphorylation detected at 15 min but not following 120 min of
exposure to either ADP or 2-MeSATP, even at the highest concentration
tested (Fig. 3A). The level of ERK1 and ERK2 protein content
was consistent across the different treatment groups (Fig.
3A). A concentration dependence was again observed in the
phosphorylation of the SAPK isoforms induced by either ADP or 2-MeSATP
application for 15 min (Fig. 3B). In addition, the same
isoforms of the SAPKs were activated by both ligands, effects that were
identical to those stimulated by 2-MeSADP (Fig. 3B). Neither
ADP nor 2-MeSATP, over the concentration range tested, had any
effect on the phosphorylation status of p38 (Fig. 3B) or on
the expression levels of the proteins (Fig. 3). The phosphorylation
status of all MAP kinases was unaffected following application of the
adenosine nucleotides to wild-type astrocytoma cells (Fig.
3C).

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Fig. 3.
Effect of 2-MeSATP, ADP, or carbachol on the
phosphorylation status of the MAP kinases in astrocytoma cells.
Whole cell extracts were prepared from (A and B)
1321N1 cells heterologously expressing human P2Y1
receptors, which had been serum-starved for 4 h before incubation
with increasing concentrations of ADP or 2-MeSATP (µM)
for the times shown. C, wild-type 1321N1 cells following
serum starvation were incubated in the presence of incomplete media
(CON), 2-MeSADP (MeS, 300 nM), or
carbachol (CAR, 10 mM) for the times shown.
Changes in MAP kinase activity status were demonstrated by detection
with an antibody to the phosphorylated forms of (A and
C) ERK1 and ERK2 (ERK-P) or to (B and
C) the isoforms of the SAPK (p46-P and
p54-P shown) and p38 (p38-P) families.
Consistency of protein loading following Western analysis was
substantiated by determining the immunoreactivity of samples with
phosphorylation state independent antibodies (A, top panel
and B, left panel).
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Effect of Endogenous Muscarinic Receptors--
To determine if
endogenous Gq-coupled receptors can trigger changes in the
activities of the MAP kinases similar to those stimulated by the
recombinant P2Y1 receptor, the effect of carbachol was
examined. Native muscarinic receptors have been shown to be functionally active in 1321N1 astrocytoma cells (41). Carbachol (10 mM) induced an increase in the phosphorylation of ERK1 and ERK2 over basal levels in wild-type 1321N1 cells following exposure for
either 15 or 120 min (Fig. 3C). In addition, a marked
increase was observed in the activity of the 45-kDa SAPK isoform (Fig. 3C). However, in contrast to that evoked by 2-MeSADP in
cells stably expressing the recombinant P2Y1 receptor, the
enhanced immunoreactivity was observed at both 15 and 120 min. No
change could be detected at either time point examined in the
phosphorylation status of any of the other SAPK isoforms detected or of
p38 (Fig. 3C). Similar immunoreactive changes were observed
following carbachol treatment in the astrocytoma cells expressing the
recombinant P2Y1 receptor (data not shown). The level of
the MAP kinase protein content was consistent across the different
treatment groups in the wild-type astrocytoma cells and showed an
expression pattern that was identical to that observed in the
transfected line (Fig. 3).
2-MeSADP Concentration-Response Relationship and Antagonism of the
P2Y1 Receptor-mediated Phosphorylation of MAP
Kinases--
The concentration dependence of the phosphorylation
changes induced by 2-MeSADP at the human recombinant P2Y1
receptor was determined at the peak of the transient response (15 min)
and also during the sustained phase of the MAP kinase activity profiles (2 h). Enhanced immunoreactivity was observed using the
anti-phosphospecific ERK antibody, in samples from 1321N1 cells
incubated for 15 min with increasing concentrations of 2-MeSADP, until
a maximal response was obtained at 30 nM (Fig.
4A). At 120 min (sustained
phase), the phosphorylation of ERK1 and ERK2 also increased in a
concentration-dependent manner, with a maximal response at and
above 100 nM 2-MeSADP (Fig. 4B). In contrast,
the phosphorylation of the SAPK isoforms at 15 min continued to
increase over the entire concentration range of 2-MeSADP used (1-1000
nM) (Fig. 5A).
Phosphorylation of p38 and the 48-kDa isoform of SAPK remained
unchanged over basal levels following incubation for 15 min with
increasing concentrations of 2-MeSADP (up to 1 µM). In
addition, no changes were observed in the phosphorylation states of p38
or any of the SAPK isoforms at 120 min over the concentration range of
2-MeSADP examined (Fig. 5B). At all 2-MeSADP concentrations
used in this study the level of expression of the MAP kinases was
unaffected (Figs. 4 and 5).

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Fig. 4.
Antagonism of the 2-MeSADP-mediated
phosphorylation of ERK1 and ERK2 in astrocytoma cells heterologously
expressing P2Y1 receptors. Whole cell extracts were
prepared from serum-starved cells (for 4 h) that had been
incubated with and without the P2Y1 receptor-selective
antagonist, A2P5P (10 µM), for 30 min before 15-min
(A) or 120-min (B) incubation with increasing
concentrations of 2-MeSADP (nM). Consistency of protein
loading was substantiated by determining the immunoreactivity of
samples with phosphorylation state independent anti-ERK antibodies
(top panels). Phosphorylation changes were demonstrated by
detection with an antibody to ERK1 and ERK2 that recognizes only the
dually phosphorylated and hence active forms (ERK-P).
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Fig. 5.
Antagonism of the 2-MeSADP-mediated
phosphorylation of SAPKs in astrocytoma cells heterologously expressing
P2Y1 receptors. Whole cell extracts were prepared from
serum-starved cells that had been incubated with and without A2P5P (10 µM) for 30 min before 15-min (A) or 120-min
(B) incubation with increasing concentrations of 2-MeSADP
(nM). Consistency of protein loading was substantiated by
determining the immunoreactivity of samples with phosphorylation state
independent antibodies to p38 and the SAPKs (top panels).
Phosphorylation changes were demonstrated by detection with an antibody
to p38 that recognizes only the dually phosphorylated (at
Thr180 and Tyr182) and hence active forms
(p38-P). Similarly, SAPK activation was assessed using an
antibody specific for the doubly phosphorylated forms of all SAPK
isoforms at residues Thr183 and Tyr185 within
the TPY sequence.
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To confirm that changes in the phosphorylation state of the MAP kinases
observed upon application of 2-MeSADP were due to the activation of the
heterologously expressed P2Y1 receptor, the transfected
cells were preincubated with the P2Y1 receptor-specific antagonist, A2P5P (10 µM), for 30 min. Phosphorylation of
ERK1 and ERK2 induced by a 15-min incubation with 2-MeSADP was
abolished over the agonist concentration range 1-100 nM in
cells pretreated with the antagonist and partially inhibited when using
the agonist at concentrations of 300 nM or greater (Fig.
4A). The activation of the SAPK isoforms induced by 2-MeSADP
at 15 min was similarly abolished by pretreatment with A2P5P (10 µM) at all concentrations of agonist tested (Fig.
5A). The activity of the 48-kDa SAPK isoform and of p38 was
unaffected by treatment with the P2Y1 receptor-selective antagonist. Phosphorylation of ERK1 and ERK2 induced by incubation with
2-MeSADP (1-1000 nM) for 120 min was also greatly
attenuated in cells pretreated with A2P5P (Fig. 4B), whereas
basal phosphorylation of p38 and all the SAPK isoforms observed at this
time point remained unchanged (Fig. 5B). The antagonist had
no effect on the expression levels of the MAP kinases or on their basal
phosphorylation status at the times investigated (Fig.
6).

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Fig. 6.
Effect of the antagonist, A2P5P, on the basal
activity status of MAP kinases in astrocytoma cells heterologously
expressing P2Y1 receptors. Whole cell extracts were
prepared from serum-starved astrocytoma cells (for 4 h) that had
been treated in the absence (CON) or presence of A2P5P (10 µM) for 30 min before a further 15- or 120-min incubation
with incomplete media. Consistency of protein loading was substantiated
by determining the immunoreactivity of samples with phosphorylation
state independent antibodies to ERK1 and ERK2 (A) or the
SAPKs and p38 (top panels) (B). Detection with
the phospho-specific antibodies is shown in the lower panels
following long exposure times of the autoradiographic film.
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Effect of MEK1, PKC, Src, and PI 3-K Inhibitors on the Induced
Phosphorylation of ERK1 and ERK2 by P2Y1 Receptors
Expressed in 1321N1 Cells--
The effect of selective inhibitors of
MEK1 (PD 98059, 20 µM), Src (PP1, 200 nM), PI
3-K (LY 294002, 100 µM), or PKC (Gö 6976 for
Ca2+-dependent isoforms, 10 nM, and
Ro 32-1432 for all isoforms, 50 nM) on the phosphorylation
of ERK1 and ERK2 induced by 2-MeSADP (50 nM) was examined.
Preincubation of the cells with these inhibitors for 10 min had no
observable effect on basal levels of ERK phosphorylation obtained at 15 min (Fig. 7A). The
2-MeSADP-induced phosphorylation at 15 min was greatly inhibited by
pretreatment with the PI 3-K or Src inhibitors as well as by either of
the PKC inhibitors. PD 98059 at the concentration used was less
effective in reducing the 2-MeSADP-induced phosphorylation (Fig.
7A). Basal levels of ERK phosphorylation observed at 120 min
were unaffected by preincubation with LY 294002, PP1, or PD 98059 (Fig.
7B), whereas that induced by 2-MeSADP was abolished by LY
294002 and PD 98059 but unaffected by PP1 (Fig. 7B). In
contrast, both protein kinase C inhibitors increased basal levels of
ERK phosphorylation, comparable to that observed following activation
by 2-MeSADP (Fig. 7B). This enhanced basal activity of ERK1
and ERK2 after a 2-h period in the presence of the PKC inhibitors was
also evident in non-transfected astrocytoma cells (data not shown). The
expression levels of ERK1 and ERK2 were unaffected by the kinase
inhibitors at both time points examined (Fig. 7).

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Fig. 7.
Effect of various kinase inhibitors on
the 2-MeSADP-mediated phosphorylation of ERK1 and ERK2 in astrocytoma
cells heterologously expressing P2Y1 receptors. Whole
cell extracts were prepared from serum-starved astrocytoma cells (for
4 h) that had been treated with the following inhibitors for 10 min before 15-min (A) or 120-min (B) incubation
with 2-MeSADP (MeS, 50 nM) or incomplete media
(Basal): LY 294002 (LY, 100 µM), PD
98059 (PD, 20 µM), PP1 (200 nM),
Gö 6976 (Gö, 10 nM) or Ro 32-1432 (Ro, 50 nM). Basal phosphorylation without
inhibitors present is shown (CON). Consistency of protein
loading was substantiated by determining the immunoreactivity of
samples with phosphorylation state independent anti-ERK antibodies
(top panel). Phosphorylation changes were demonstrated by
detection with an antibody to ERK1 and ERK2 that recognizes only the
dually phosphorylated and hence active forms (ERK-P).
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Effect of a Dominant Negative Mutant of Ras or Pertussis Toxin
Pretreatment on P2Y1 Receptor-mediated ERK
Phosphorylation--
To evaluate the involvement of Ras in mediating
the activation of ERK by P2Y1 receptors, transient
expression of the dominant negative mutant of
RasAsn-17 was performed. This Ras mutant, in which
amino acid 17 (serine) is changed to asparagine, is thought to function
by inhibiting guanine nucleotide exchange factors (42). The increase in
RasAsn-17 levels following transfection was
evaluated by immunoblotting cell extracts immediately prior to drug
addition with a polyclonal antibody to Ras. The inset in Fig.
8B shows that in
mock-transfected cells the immunoreactivity with the anti-Ras antibody
was almost undetectable, compared with the intense reactivity obtained
from the same number of cells transfected with pUSEamp(+) plasmids containing dominant negative Ha-RAS. The transfection
efficiency for all experiments was between 46 and 52%. The level of
phosphorylation of ERK1 and ERK2 induced by a 15- or 120-min
application of 2-MeSADP (50 nM) was unchanged by
transfection with the empty plasmid (Fig. 8, A and
B). However, following transient expression of
RasAsn-17, the 2-MeSADP-induced phosphorylation at
15 min (Fig. 8A) was decreased but unaffected at 120 min
(Fig. 8B). Neither transfection with pUSEamp or
pUSEamp(RASAsn-17 showed any effect on basal levels
of ERK phosphorylation observed at 15 or 120 min (Fig. 8, A
and B). To show consistency in protein loading, detection of
ERK1 and ERK2 using phosphorylation state-independent pan antibodies
was also performed (Fig. 8, A and B).

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Fig. 8.
Effect of dominant negative Ras or pertussis
toxin on the phosphorylation of ERK1 and ERK2 and the effect of
dominant negative MKK4 on SAPK activation in cells expressing
P2Y1 receptors. Whole cell extracts were prepared from
1321N1 cells that had been transiently transfected either with the
empty plasmid (Mock) or the plasmid incorporating dominant
negative Ha-RAS RASAsn-17 48 h prior
to incubation for 15 min (A) or 120 min (B) in
incomplete media (CON) or 2-MeSADP (MeS, 50 nM). The effect of these agents on the phosphorylation
status of ERK1 and ERK2 (ERK-P) in non-transiently
transfected cells is also shown. All cells were serum-starved for
4 h prior to the application of 2-MeSADP. Consistency of protein
loading was substantiated by the regular intensity of the
immunoreactivity obtained following detection of the samples shown with
the anti-ERK antibodies (ERK). The Western blots are representative of
two separate transfections, and each panel has been taken from a single
immunoblot. The inset shows an immunoblot of protein from
cell samples extracted immediately prior to drug addition that had been
transiently transfected with either pUSEamp (Mock) or
pUSEamp incorporating dominant negative Ha-RAS
(RASAsn-17). Following separation by 15%
polyacrylamide gel electrophoresis and transfer onto nitrocellulose,
detection was made with an anti- -actin antibody to demonstrate
consistency of protein loading (not shown) as well as with an antibody
to Ras. C, whole cell protein extracts were prepared from
1321N1 cells incubated for 15 min with incomplete media (CON) or
2-MeSADP (MeS, 50 nM) following pertussis toxin
pretreatment (PTX, 18 h at 100 ng
ml 1) and analyzed by Western blotting using
phospho-specific anti-ERK antibodies (ERK-P). D,
whole cell extracts were also prepared from cells that had been
transiently transfected either with the empty plasmid (Mock)
or that incorporating dominant negative MKK4 48 h prior to
incubation for 15 min in incomplete media (CON) or 2-MeSADP
(MeS, 300 nM). The effect of these agents on the
phosphorylation status of the SAPKs and p38 is shown together with data
from non-transiently transfected cells. The Western blot is a
representative from two separate transfections. The inset
shows the level of expression of MKK4 immediately prior to drug
addition.
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Preincubation of the clonal 1321N1 cells with pertussis toxin (100 ng
ml
1 for 18 h) had no observable effect
on basal levels of ERK phosphorylation obtained at 15 min (Fig.
8C). The 2-MeSADP-induced phosphorylation (50 nM) was also unaffected by treatment with the toxin (Fig. 8C). The level of expression of ERK1 and ERK2 protein was
unchanged by pertussis toxin pretreatment (Fig. 8C).
Effect of a Dominant Negative Mutant of MKK4 on P2Y1
Receptor-mediated SAPK Phosphorylation--
SAPKs are activated by
phosphorylation on threonine and tyrosine within the activation motif
by one of two cloned dual specificity kinases, MKK4 and MKK7. These
kinases are in turn activated by an MKKK, of which several examples
have been identified. Transient expression (54% efficiency) of a
dominant negative mutant of MKK4 in astrocytoma cells was found to
decrease the levels of SAPK phosphorylation induced by a 15-min
application of 2-MeSADP (300 nM), compared with
mock-transfected controls (Fig. 8D). However, the
suppression of the activity status of the higher molecular weight SAPK
species appeared to be greater than for the isoform with apparent
molecular mass of 45 kDa (Fig. 8C). Expression of the mutant
MKK4 protein was determined by immunoblotting the transient transfected
cell extracts immediately prior to 2-MeSADP addition. The
immunoreactivity with the anti-MKK4 antibody was weak in
mock-transfected cells compared with the intense reactivity obtained
from the same number of cells transfected with pCMV-MKK4(K95R)
(inset, Fig. 8D). The pattern of 2-MeSADP-induced
SAPK phosphorylation was similar in cells containing the empty plasmid
compared with those that had not been transiently transfected (Fig.
8D). Transfection with either plasmid showed no apparent
effect on basal levels of SAPK phosphorylation observed at 15 min (Fig.
8D).
Regulation of Transcription Factors by the Human P2Y1
Receptor--
Phosphorylation of transcription factors is a
prerequisite for their activation (43). By using phospho-specific
antibodies, the regulation of c-Jun, activating transcription factor-2
(ATF-2) and Elk-1 by the recombinant P2Y1 receptor, was
determined using Western analysis of whole cell extracts from the
P2Y1 receptor-expressing cell line. Phosphorylation of the
transcription factors c-Jun and Elk-1 was detectable under basal
conditions following incubation with incomplete media and remained
unchanged over the time course investigated (Fig.
9A). There was only faint
immunoreactivity detectable at all time points examined under basal
conditions using antibodies to phosphorylated ATF-2. Incubation with
2-MeSADP (300 nM) had no effect on the basal
immunoreactivity detected for c-Jun or ATF-2 over the time course
investigated (Fig. 9A). However, 2-MeSADP increased the
phosphorylation of Elk-1 over time, reaching a maximal response
following 60 min of agonist application and which was maintained
throughout the duration of the time course (Fig. 9A). The
level of expression of the transcription factors was unaffected by the
application of 2-MeSADP and unchanged over the period investigated
(data not shown). The phosphorylation of Elk-1 induced by 2-MeSADP at
60 min was inhibited by a 10-min preincubation with PD 98059 (20 µM). Basal levels of Elk-1 phosphorylation at this time
point were also slightly inhibited by the MEK1 inhibitor (Fig.
9B). The expression levels of Elk-1 were unaffected by all treatments (Fig. 9B).

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Fig. 9.
Effect of 2-MeSADP on the phosphorylation of
transcription factors in astrocytoma cells heterologously expressing
P2Y1 receptors. A, whole cell extracts were
prepared from serum-starved cells (T0) that had
been incubated in the presence of incomplete media (Basal)
or 2-MeSADP (300 nM) for the times shown (minutes).
Activation of the transcription factors Elk-1, c-Jun, and ATF-2 was
determined using antibodies that recognize the phosphorylated forms of
the proteins. B, samples were analyzed for Elk-1 expression
(left panel) or with an antibody recognizing the
phosphorylated form of Elk-1 (right panel) from cells
preincubated with or without PD 98059 (PD, 20 µM) for 10 min before a 60-min incubation with either
incomplete media (CON) or 2-MeSADP (MeS, 300 nM).
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Induction of Apoptosis but Not Cell Proliferation by the Human
Recombinant P2Y1 Receptor and Effect of Ras and MKK4
Dominant Negative Mutants--
Following treatment for 5 h with
either 2-MeSADP (300 nM) (Fig.
10A) or staurosporin (300 nM) (data not shown), some astrocytoma cells fluoresced
brightly in the presence of annexin V-FITC which were not
counterstained by propidium iodide. In these cells FITC green
fluorescence patches could be observed by confocal microscopy that were
located on the cell surface (Fig. 10, B and C).
None of the control cells incubated in media alone showed any
detectable annexin V binding at this time point (data not shown).
Activation of caspase-3 following stimulation of P2Y1
receptors was also determined. Caspase-3 activity within cell lysates
(measured by the hydrolysis of a colorimetric substrate peptide)
increased as a function of time following treatment of cells with
2-MeSADP (300 nM) and was significantly above that in
samples incubated for 3 h and more with media alone (Fig.
10D). In addition, activation of caspase-3 activity by
2-MeSADP was attenuated by A2P5P (10 µM) (Fig.
10D). A2P5P had no effect on basal caspase activity (data not shown).

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Fig. 10.
Effect of 2-MeSADP on annexin V
binding, caspase-3 activation, and the proliferation of astrocytoma
cells heterologously expressing P2Y1 receptors.
Annexin V binding to astrocytoma cells expressing human
P2Y1 receptors following incubation for 5 h with
2-MeSADP (300 nM) as detected by low power fluorescence
microscopy (scale bar, 50 µm) (A) or confocal
microscopy (B and C) which shows a cluster of
annexin V-positive cells (green) in the center of the field
of the propidium iodide-stained cells (red), where
B is a single channel overlay confocal image (scale,
146.2 × 146.2 µm), and C is a single overlay of a
group of apoptotic cells taken from z series (scale, 59 µm × 59 µm). D and E, in vitro
caspase-3-like protease activity was determined using a colorimetric
caspase-3 substrate peptide. D, cell lysates were prepared
at the indicated times after incubation of astrocytoma cells in
incomplete media (Basal, open histograms),
2-MeSADP (300 nM, closed histograms), or
2-MeSADP (300 nM) following a 30-min preincubation with
A2P5P (10 µM, hatched histograms). Values are
expressed in arbitrary units as the mean ± S.E.
(n = 3). Groups labeled * are significantly different
from basal (p < 0.001) and groups labeled # are
significantly different from those incubated with 2-MeSADP alone.
E, caspase-3 activity was determined 5 h following
incubation in incomplete media (Basal, open
histograms) or 2-MeSADP (300 nM, closed
histograms) with or without a 10-min preincubation with inhibitors
of Src (PP1, 200 nM), PI 3-K (LY
294002, 100 µM), MEK1 (PD 98059, 20 µM), or the classical PKC isoforms (Gö
6976, 10 nM). Data are also shown for cells
transfected 48 h prior to drug addition with either dominant
negative Ras or MKK4 mutants. Groups labeled * are significantly
different from that without inhibitors present (p < 0.01), and the group labeled # is significantly different from the same
treatment but in non-transiently transfected cells. F, the
mean number of cells harvested from a single well, 24 h following
application of incomplete media (Basal, open
histogram) or 2-MeSADP (300 nM, closed
histogram). Groups labeled mock had been transfected
48 h previously with an empty vector, and those labeled
MKK4 had been transfected with the vector containing the
kinase-deficient mutant of MKK4. Values are expressed as the mean cell
number ± S.E. (n = 2, four replicates). The group
labeled * is significantly different from basal (p < 0.01). G, the mean number of cells harvested from a single
well, 24 h following application of incomplete media
(Basal, open histogram), 2-MeSADP (300 nM, closed histogram), fetal calf serum
(FCS, 1%; hatched histograms), 2-MeSADP (at 0.3 or 3 µM) in the presence of 1% serum (MeS + FCS, shaded histograms), carbachol (10 mM,
striped histogram) or 2-MeSADP (300 nM) in the
presence of carbachol (MeS + CAR, crossed
histogram). Values are expressed as the mean cell number ± S.E. (n = 3, four replicates). Groups labeled * are
significantly different from basal (p < 0.01) and that
labeled # is significantly different (p < 0.01) from
that incubated in the presence of carbachol alone.
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The effects of selective inhibitors and of the dominant negative
mutants of Ras and MKK4 were also determined on the ability of 2-MeSADP
to induce caspase-3 activity. Astrocytoma cells expressing the
recombinant P2Y1 receptor were pretreated for 10 min with PD 98059 (20 µM), PP1 (200 nM), LY 294002 (100 µM), or Gö 6976 (10 nM) before a
further 5-h incubation with or without 2-MeSADP (300 nM)
present. Basal caspase-3 activity and that induced by 2-MeSADP at this
time point were unaffected by PPI, PD 98059, or Gö 6976 but were increased slightly in the presence of the PI 3-K inhibitor
(Fig. 10E). Transfection of cells (48 h prior to the
addition of 2-MeSADP) with dominant negative forms of either Ras or
MKK4 had no significant effect on basal levels of caspase-3 activity
compared with mock-transfected cells (data not shown) and were similar
to the values obtained for non-transfected cells expressing the
P2Y1 receptor (Fig. 10E). Dominant negative Ras expression also had no effect on the caspase-3 activity induced by
2-MeSADP, whereas the kinase-deficient MKK4 mutant partially blocked
this latter response (Fig. 10E).
The ability of 2-MeSADP to modulate the proliferative outcome of
astrocytoma cells expressing the recombinant P2Y1 receptor was also assessed. Application of 2-MeSADP (300 nM)
in the absence of other exogenously administered mitogenic
factors showed a small but significant decrease in the number of cells
counted 24 h later, as compared with basal, which was not observed
in cells transiently transfected with the MKK4 mutant (Fig.
10F). In contrast, fetal calf serum (1%) caused a
significant increase in cell number (Fig. 10G) that was
unaffected by the addition of 2-MeSADP at concentrations of 0.3 or 3 µM (Fig. 10G). A marked increase in cell
number was also obtained following application of carbachol (10 mM) that was attenuated by the presence of 2-MeSADP (300 nM) (Fig. 10G).
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DISCUSSION |
In this study, we have examined the ability of the human
P2Y1 receptor to stimulate the MAP kinase transduction
cascades and to determine if this activity could be correlated with
transcription factor phosphorylation or the proliferative outcome of
the host cell. Whereas a stably transfected cell line was employed for these studies, the host cell was derived from a human type
(astrocytic), which in its original parent state expressed functional
P2Y1 receptors (18) and hence should contain the
appropriate native transductional components for the recombinant
receptor. The presence of P2Y1 as the sole P2Y receptor has
enabled the transduction cascades of an identified receptor type to be
characterized. Definitive receptor specificity was also confirmed by
the suppression of the stimulated responses by a P2Y1
receptor-selective antagonist. Expression of the P2Y1
receptor protein at the cell membrane of the transfected cells was
demonstrated by immunocytochemistry, using an anti-peptide antibody
specific for the human P2Y1 receptor subtype. The size of
the immunoreactive band detected in Western blots of the astrocytoma
cells is consistent with the known polypeptide size of the
P2Y1 receptor (373 amino acids, 42 kDa) and its
glycosylation (1).
Functional responses of the P2Y1 receptor have been shown
to be mediated through G11 and/or Gq proteins,
which were shown to be expressed by the astrocytoma host cells used in
this study. Upon agonist stimulation, G protein-coupled receptors
transduce their effects through both the GTP-bound G
and the
dissociated G
component of the heterotrimeric G protein,
regulating directly downstream effectors (44) including adenylate
cyclases, phospholipase C isoforms, ion channels, PI 3-K (45), and Tec
family tyrosine kinases (46). Several G protein-coupled receptors have
been shown to stimulate the ERK pathway through a variety of G protein subunits. In the case of the Gq/11-coupled m1
muscarinic acetylcholine and
1-adrenergic receptors, the
activation of ERK is mediated mainly by G
q/11. In
contrast, Gi-coupled m2 muscarinic
acetylcholine,
2-adrenergic, somatostatin
sst2, and the Gs-coupled
-adrenergic receptors, all induce ERK activation through G
release and the subsequent stimulation of tyrosine kinases such as Src (47, 48). The
pathways downstream of G protein coupling have not previously been
established for any molecularly defined P2Y receptor subtype. In the
present report, the potential actions of nucleotides at the
P2Y1 receptor in stimulating the MAP kinase cascades were investigated. The P2Y1 receptor had no effect on the
activity status of p38 kinase but caused a marked phosphorylation of
the SAPK and the ERK cascades. For the ERK cascade, this was shown to
be via a pertussis toxin-insensitive pathway through a number of
transduction mediators including PKC, PI 3-K, and Src, with apparent
cooperative effects.
The mechanisms by which Gi- and
Gq-coupled receptors typically activate ERK are through
Ras-dependent or protein kinase C-dependent pathways, respectively. However, several exceptions to this rule have
been reported for Gq-coupled receptors, in that ERK can be activated through a pertussis toxin-insensitive and PKC-independent pathway (49). The recombinant P2Y1 receptor utilizes
transduction cascades to activate ERKs that are thus commonly
associated with Gq-linked receptors (PKC activation) in
addition to the recruitment of Src implying some Ras dependence, which
is more typical of Gi-coupled receptors. An involvement of
Ras in the mediation of the P2Y1 receptor-induced ERK
activity was demonstrated in this study by overexpression of a dominant
negative Ras mutant. However, although inhibition of Src or Ras
attenuated the transient phase of the ERK activity profile, these
transduction effectors did not seem necessary for the sustained
activation of ERK. This differential requirement for transduction
mediators during the time course of ERK activation has also recently
been demonstrated for the Gi/o-coupled somatostatin
sst4 receptor in which the acute phase is both Src- and
Ras-dependent, but the prolonged ERK response is mediated
by protein kinase C (50). The mobilization of Ca2+ through
the P2Y1 receptor could account for the stimulation of the
Ca2+-dependent PKC isoforms that have been
shown to activate the ERK cascade at the point of Raf (51) and thus
through a Ras-independent mechanism.
The PI 3-K pathway is also important for regulating ERK activity
by a number of mechanisms that have been shown to occur both upstream
and downstream of Ras (52, 53). The sustained phase of ERK
phosphorylation mediated through the P2Y1 receptor was abolished following application of a PI 3-K inhibitor, whereas the
transient phase was only partially dependent on this kinase activity.
It thus appears that the transient activation of ERK by the
P2Y1 receptor is mediated through the cooperative effects of Src, Ras, Ca2+-dependent PKC isoforms as
well as PI 3-K, whereas the sustained phase requires only PI 3-K
activity (and possibly PKC). This mechanism may be similar to that
reported for Gi protein-coupled receptors that have been
shown to activate ERK via a Ras-independent pathway through PI 3-K
and PKC
(54). Stimulation of PKC isoforms in addition to their
requirement for allosteric activators has recently been shown to be
critical on subsequent phosphorylation, possibly through the PI
3-K-dependent kinase, PDK1 (55). In accord with this
concept is the finding that ERK phosphorylation by the P2Y1 receptor requires PI 3-K for both the acute and sustained phases. We
could not conclude if the sustained phase of ERK activation is
additionally mediated via PKC due to the marked increase in basal ERK
activity observed in the presence of various PKC inhibitors. The cause
of this ERK stimulation following prolonged PKC down-regulation has not
been determined but could also be observed in non-transfected astrocytoma cells. The ineffectiveness of the MEK1 inhibitor at blocking the P2Y1 receptor-mediated transient activation of
ERK1 and ERK2 may reflect the inability of PD 98059 to abolish high intensity signals (56). However, PD 98059 is a MEK1-selective inhibitor, and it is possible that ERK activation through
P2Y1 receptors is primarily mediated through MEK2 in the
early transductional events and via MEK1 for the sustained phase of stimulation.
The rapid and sustained phosphorylation of both ERK1 and ERK2 induced
by the potent P2Y1 receptor agonist, 2-MeSADP, was
abolished by the P2Y1 receptor-specific antagonist, A2P5P.
The concentration dependence of this ERK activity was similar to that
observed with 2-MeSADP for other functional responses mediated by this
receptor type (EC50 values of the order of 10 nM) (9-11, 57). The natural ligand ADP, although less
potent than 2-MeSADP, induced phosphorylation of ERK1 and ERK2 that was
comparable to that evoked by 2-MeSATP, supporting recent evidence that
uncontaminated adenosine triphosphates can serve as agonists at the
recombinant P2Y1 receptor (10, 11). Release of ATP by cells
in culture occurs very readily (1) and can also give rise to misleading
results; this might have contributed to the transient and low degree of
ERK1 and ERK2 stimulation observed in this study following incubation
of the astrocytoma cells with incomplete media. However, the
P2Y1 receptor-selective antagonist, A2P5P, showed no effect
on the basal levels of ERK phosphorylation, suggesting that this
activity may result directly from mechanical stimulation (34).
Although the Ras-ERK cascade is well documented for several G
protein-coupled receptors, very little is known of any activation of
the other MAP kinases, p38 and the SAPKs, by this receptor class.
Activation of p38 has been shown in rat glomerular mesangial cells
following stimulation with UTP and ATP, suggesting that this may be
mediated through the P2Y2 receptor (27). In addition, a
very recent report on native P2Y receptors in the endothelial cell
line, EAhy926, has shown an unusual inhibition of a pre-stimulated SAPK
and p38 activity mediated by UTP and suggested to be via P2Y2 or P2Y4 receptors (58). In the present
study, we have shown differential activation of members of the SAPK
family through the P2Y1 receptor but not of p38. The
kinetic profiles of the SAPK isoforms activated, those with apparent
molecular masses of 45, 46, and 54 kDa, were similar and transient,
although a fourth immunoreactive band of 48 kDa that was identified by
both the phospho-dependent and -independent SAPK antibodies
remained unaffected by 2-MeSADP, ADP, or 2-MeSATP. Phosphorylation of
the p54, p46, and p45 SAPK isoforms was inhibited by the
P2Y1 receptor-specific antagonist, A2P5P. At least 10 SAPK
isoforms have been identified that correspond to alternatively spliced
isoforms derived from the JNK1, JNK2, and
JNK3 genes (59). The consequence of the differential
activation of the SAPK isoforms identified in the astrocytoma cells by
the P2Y1 receptor remains to be determined, but possibly
provides fine-tuning of various cellular events including apoptosis (see below). In addition, this differential activation suggests that discrete mechanisms are in place upstream of the individual SAPK family members. Stimulation of only the p45 form of the
SAPKs by the muscarinic receptor agonist, carbachol, and the finding
that this displayed a different kinetic profile to that induced by the
P2Y1 receptor provide additional evidence that members of
this MAP kinase family are regulated via distinct mechanisms in the
same host cell.
Proximally, SAPKs are activated by a cascade of kinases (17), although
the upstream regulators in this pathway are incompletely characterized.
Tumor necrosis factor-
-stimulated SAPK activation is perhaps best
described and involves recruitment of the adapter protein TRAF2 to the
cytosolic portion of the ligated tumor necrosis factor-
receptor
(60). Other intermediates have been proposed to play a role in
different models of SAPK activation including oxidative stress, DNA
damage, altered ion fluxes, and caspase proteases. However, the present
study suggests that caspase-3 activation is not an obligatory step in
the signaling pathway coupling P2Y1 receptors to SAPK
activation, as this MAP kinase activity preceded the accumulation of
active caspase-3 by several hours. It is possible that changes in
intracellular Ca2+ (61) may be important for the
P2Y1 receptor-mediated SAPK activation. SAPKs are activated
by phosphorylation on threonine and tyrosine residues by one of two
cloned dual specificity kinases, MKK4 and MKK7. The dependence on MKK4
for the activation of the p54 and p46 SAPK isoforms mediated by the
P2Y1 receptor was substantiated by showing a decrease in
their phosphorylation status following expression of a kinase-deficient
MKK4 mutant. The induced phosphorylation of the p45 isoform by 2-MeSADP
was only slightly affected by the MKK4 mutant, which may possibly
reflect the achieved transient transfection efficiency or that MKK7 may
be the preferred upstream regulator of this kinase.
The apparent lack of phosphorylation of c-Jun and of ATF-2
transcription factors, both known substrates for the SAPKs, suggests that the observed transient activation of these kinases, induced via
the recombinant P2Y1 receptor, is insufficient for
their cytoplasmic nuclear translocation. In every case studied so far,
sustained ERK activation is required for nuclear-targeted transcription factor phosphorylation, although similar observations for the SAPK
family have not as yet been reported. Consistent with this hypothesis
is the demonstration that sustained ERK activity induced through the
P2Y1 receptor produced Elk-1 phosphorylation. These data,
taken together, thus suggest that the strength and duration of the
stimulus to other MAP kinase family members can also be important
determinants that govern the biological response to a particular
receptor activation.
The functional responses mediated by events downstream from the
activation of P2Y receptors are poorly understood. It has been
suggested that prostacyclin production following activation via P2Y
receptors on endothelial cells is via ERK activation (20), consistent
with the mechanism utilized by endothelial cells to regulate vascular
smooth muscle cell proliferation. Proliferative activity has been
reported using UTP in C6 glioma cells through P2Y2 receptor-mediated stimulation of the Ras-ERK pathway
(28). P2Y4 receptors in rat glomerular mesangial cells have
been shown to induce proliferation (29), whereas stimulation via the
P2X7 receptor induces apoptosis (32). ERK is almost
universally stimulated by mitogens and cell survival factors such as
growth factors, hormones, and cytokines and is intimately connected
with the regulation of cell growth as well as differentiation. SAPK and
p38 on the other hand are activated by various stressors such as
chemical agents and ultraviolet irradiation, tumor necrosis factor, and interleukin-1, which appear to play a decisive role in the control of
cell death. A necessary role of SAPK in apoptotic induction by UV
irradiation, but not Fas receptor ligation, was demonstrated in a
recent study using embryonic fibroblasts derived from double knockout
mice that lack the expression of both the JNK1 and
JNK2 genes (62). It has been suggested that the ability of a
cell to die or survive and proliferate may be dictated by a critical balance between the signaling pathways involving the various MAP kinase
family members.
In the present study, we have shown that despite the sustained
activation of ERK1 and ERK2 by the P2Y1 receptor, a
proliferative activity was not apparent. This is analogous to the
situation with the somatostatin sst2(a) receptor, which
mediates a strong and sustained activity of both ERK1 and ERK2 but that
is associated with a concomitant anti-proliferative effect (48).
However, activation of the endogenous muscarinic receptors in the
astrocytoma cells induced a sustained ERK activation and an associated
increase in cell number, suggesting that the proliferative outcome of
this cell type is regulated by a complex interplay of transduction cascades. A role for the SAPK family members activated by the P2Y1 receptor in the induction of an apoptotic event was
confirmed by showing that the 2-MeSADP-stimulated caspase-3 activity
and the associated decrease in cell number was inhibited by the
presence of the dominant negative MKK4 mutant. The partial nature of
the decrease in both cases could reflect the degree of transient
transfection efficiency obtained (~50%). However, it would seem that
the p54 and p46 isoforms are much more important for apoptotic function than the p45 SAPK member, as the activity of this latter isoform was
relatively unaffected by the MKK4 mutant and was strongly increased by
the application of carbachol. The P2Y1 receptor-induced caspase-3 activity, which could be blocked by the selective antagonist, A2P5P, appeared to be unaffected by inhibitors of the Ras-ERK cascade
(including the Src inhibitor and a dominant negative Ras mutant). This
suggests that there is little cross-talk between the ERK and SAPK
cascades activated by the P2Y1 receptor. The role of PI 3-K
in regulating the apoptotic function of 2-MeSADP is less clear, as LY
294002 enhanced the basal rate of caspase-3 activity as well as that
mediated by the P2Y1 receptor. It is possible that the PI
3-K cascade is having an additional effect on maintaining cell survival
that is independent of SAPK inhibition and presumably of its ability to
induce ERK phosphorylation. Interestingly, the P2Y1
receptor-mediated apoptotic activity was not sufficient to inhibit cell
proliferation induced by 1% serum, despite high concentrations of
2-MeSADP being used to circumvent any problems associated with ligand
depletion. It is possible that the transient activation of the SAPK
isoforms through P2Y1 receptor activation may be to
regulate apoptotic events rather than to induce cell death as shown for
the P2X7 receptor that mediates sustained activation of the
SAPKs in the presence of serum (32). However, the proliferative activity induced by carbachol was attenuated by the presence of 2-MeSADP, further suggesting that its ability to induce apoptosis is
dependent on the net activity of transductional cascades.
In summary, we have demonstrated that the P2Y1 receptor can
stimulate the prolonged activation of the ERK cascade leading to the
phosphorylation of the transcription factor, Elk-1. This sustained ERK
activity is critically dependent on PI 3-K, whereas the transient phase
is mediated through Ras with an additional input from both PI 3-K and
PKC isoforms. Transient activation of the SAPKs in this system is
insufficient for transcription factor phosphorylation but appears to
regulate apoptosis through a caspase-3-dependent mechanism.
Hence, an identified member of the P2Y receptor family can activate
SAPKs (but spares p38 kinase) and can evoke caspase proteolytic
activity. This duality suggests that cell proliferation by
extracellular nucleotides may be regulated by a critical balance of the
activities of those receptor types, which mediate mitogenic or
apoptotic processes.