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1 Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65212; 2 Research Service, Veterans Affairs Medical Center, Departments of Pathology, Biochemistry and Molecular Biology, and the Neuroscience Program, University of Miami School of Medicine, Miami, Florida 33125; and 3 Department of Chemistry, University of Puerto Rico, Rio Piedras, Puerto Rico 00931
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Astrocytes are involved in normal and
pathological brain functions, where they become activated and undergo
reactive gliosis. Astrocytes have been shown to respond to
extracellular nucleotides via the activation of P2 receptors, either G
protein-coupled P2Y receptors or P2X receptors that are ligand-gated
ion channels. In this study, we have examined the manner in which
activation of the P2X7 nucleotide receptor, an
extracellular ATP-gated ion channel expressed in astrocytes, can lead
to the phosphorylation of ERK1/2. Results showed that the
P2X7 receptor agonist
2',3'-O-(4-benzoyl)benzoyl-ATP induced ERK1/2
phosphorylation in human astrocytoma cells overexpressing the
recombinant rat P2X7 receptor (rP2X7-R), a
response that was inhibited by the P2X7 receptor
antagonist, oxidized ATP. Other results suggest that
rP2X7-R-mediated ERK1/2 phosphorylation was linked to the
phosphorylation of the proline-rich/Ca2+-activated tyrosine
kinase Pyk2, c-Src, phosphatidylinositol 3'-kinase, and protein
kinase C
activities and was dependent on the presence of
extracellular Ca2+. These results support the hypothesis
that the P2X7 receptor and its signaling pathways play a
role in astrocyte-mediated inflammation and neurodegenerative disease.
astrocytes; P2 nucleotide receptors; ligand-gated ion channels; protein kinase C; mitogen-activated protein kinases
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ASTROCYTES PARTICIPATE in neuronal development, synaptic activity, and homeostatic control of the extracellular environment (5). Astroglial cells respond to brain injuries with reactive gliosis, characterized by astrocytic proliferation and hypertrophy (48), responses that ameliorate brain damage from injury but paradoxically contribute to neuronal cell death (4, 5). Under normal and pathological conditions, astrocytes release nucleotides (18, 45) that play a significant role in the pathophysiology of acute and chronic disorders in the central nervous system (14, 32). Moreover, nucleotide receptor mRNA has been detected in astrocytes, suggesting that astrocytic nucleotide receptors can mediate responses to nucleotides in normal and injured brains (23, 24, 27).
P2 nucleotide receptors belong to two receptor superfamilies: P2Y
G protein-coupled receptors (26, 33, 58) and P2X
ligand-gated ion channels (42). Of the seven cloned P2X
receptors, six of them (P2X1-6-R) are found in brain
and peripheral neurons. Moreover, mRNAs for P2X1,
P2X4, and P2X7 receptors are prominent in
immune and nonimmune cells (41, 42), and the
P2X7 receptor (P2X7-R) has recently been found
in neurons (6) and astrocytes (29, 43).
Activation of P2X(1-6)-R is associated
with gating of transmembrane ion channels that increase the
intracellular Ca2+ concentration, either through the
channel itself or by activation of voltage-dependent Ca2+
channels, presumably due to P2X-R-mediated
Na+/K+ conductance (9, 13, 37,
52). P2X7-R activation causes similar responses to
activation of other P2X-R, including increased membrane permeability of
ions (9, 13, 37, 52), but also unique responses such as
formation of larger pores enabling transmembrane passage of normally
membrane-impermeable molecules 
900 Da (11, 53, 55).
Moreover, formation of these pores is associated with membrane blebbing
and cell apoptosis (7, 53, 55). Recently, the
P2X7-R was shown to mediate activation of the kinases SAPK/JNK in human and rodent macrophages independent of caspase-1- or
caspase-3-like proteases (21) as well as the activation of extracellular signal-regulated kinases (ERK1/2) in rat primary astrocytes (43).
In this study, we investigated the signal transduction pathway
leading to ERK1/2 activation by the recombinant rat P2X7-R stably expressed in human 1321N1 astrocytoma cells. Results with these cell transfectants unambiguously demonstrated that the
P2X7-R mediates ERK1/2 activation dependent on the presence
of extracellular Ca2+ as well as activation of Pyk2, c-Src,
phosphatidylinositol 3'-kinase (PI 3-K), and protein kinase C
(PKC). Elucidation of this novel signal transduction pathway linking
P2X7-R stimulation to ERK1/2 activation in astrocytes could
lead to a better understanding of the pathophysiological roles for
these receptors in neurodegenerative diseases.
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MATERIALS AND METHODS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Reagents.
Dulbecco's modified Eagle's medium (DMEM) and neomycin G418 were
obtained from GIBCO BRL (Carlsbad, CA). FBS was purchased from Harlan
Bioproducts for Sciences (Indianapolis, IN). Penicillin, streptomycin,
ATP, 2',3'-O-(4-benzoyl)benzoyl-ATP (BzATP),
LY-294002, BAPTA-AM, and periodate-oxidized ATP (oATP) were
purchased from Sigma Chemical (St. Louis, MO). GF-109203X,
Gö-6976, and the c-Src inhibitor PP2 were acquired from
Calbiochem (San Diego, CA). The MEK1/2-specific inhibitor U0126, mouse
monoclonal anti-phospho-p44/p42 MAPK
(Thr202/Tyr204), rabbit polyclonal
anti-phospho-MEK1/2 (Ser217/221), rabbit polyclonal
anti-phospho-PKC (Thr505), and rabbit polyclonal
anti-MEK1/2 were purchased from Cell Signaling Technology (Beverly,
MA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG,
HRP-conjugated goat anti-rabbit IgG, rabbit anti-ERK1 (K-23), rabbit
anti-nPKC (C-17), and Western blotting Luminol reagent were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal
anti-Pyk2-p[Y402] and anti-Pyk2-p[Y881]
were purchased from BioSource International (Camarillo, CA), whereas
rabbit anti-Pyk2 was purchased from Upstate Biotechnology (Lake
Placid, NY). The RNeasy Mini kit for total RNA isolation and
purification was obtained from Qiagen (Valencia, CA). The First-Strand
cDNA Synthesis kit for RT-PCR (avian myeloblastosis virus, AMV) and the
Expand High-Fidelity PCR system were purchased from Roche
(Indianapolis, IN). All other reagents were of analytic grade or better.
Expression of P2X7 receptors and cell culture. The recombinant rat P2X7-R was expressed in human 1321N1 astrocytoma cells, as described previously for expression of the P2Y2 receptor (15, 44). Briefly, recombinant rP2X7-R cDNA incorporated in the pLXSN retroviral vector was transfected into PA317 amphotrophic packaging cells for amplification of the retroviral vectors. The pLXSN vector was used as a negative control. The 1321N1 cells were then infected with the retroviral vectors and selected for neomycin resistance with 1 mg/ml G418. Rat P2X7 receptor cDNA was obtained from GlaxoWellcome with generous assistance from Drs. Iain Chessell and Pat Humphrey (Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, Cambridge, UK). The 1321N1 cells expressing the rP2X7-R (1321N1/rP2X7) or pLXSN were cultured in DMEM containing 5% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 mg/ml G418.
RT-PCR assays of P2 receptor mRNA.
Because phospholipase C-coupled P2 receptors, especially
P2Y2, have been reported to activate ERK1/2, RT-PCR assays
were performed to determine whether the 1321N1 astrocytoma cells used
to express the recombinant rP2X7-R were devoid of mRNA to
Ca2+-mobilizing P2Y receptors. Briefly, total RNA was
isolated from human 1321N1 astrocytoma cells stably expressing the
rP2X7-R using the RNeasy Mini kit. cDNA was then
synthesized from the purified RNA by using the First-Strand cDNA
Synthesis kit for RT-PCR (AMV). Five percent of the synthesized cDNA
was used as a template in PCR by using the Expand High-Fidelity PCR
system. Oligonucleotide primers were designed to selectively amplify
P2Y receptor cDNA to specific subtypes (Table
1), as recently described
(51). The sequence-specific primers for P2X-R also were
designed to selectively amplify P2X receptor subtypes, as shown in
Table 1. P2X7 primers were based on the cDNA sequence, as
described by Rassendren et al. (46), and amplified a
728-bp sequence. The resulting PCR products were resolved on a 1%
(wt/vol) agarose gel containing 10 µg/ml ethidium bromide and were
photographed under UV illumination.
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ERK1/2, MEK1/2, and PKC phosphorylation.
Human 1321N1 astrocytoma cells stably transfected with
rP2X7-pLXSN cDNA or the pLXSN vector were grown to 85%
confluence in six-well plates. Cells were incubated in serum-free DMEM
for 18 h at 37°C before the experiments. Specified inhibitors
and/or receptor antagonists were added to the serum-free DMEM medium for the times indicated. EGTA was added as a HEPES-buffered saline solution (25 mM HEPES and 110 mM NaCl, pH 7.6) to serum-free DMEM to
give final EGTA concentrations of 0-8 mM, and cells were incubated for 5 min at 37°C. Cell transfectants were then stimulated for 5 min
at 37°C with 50 µM BzATP, a relatively specific P2X7-R
agonist (11, 16, 41), or with vehicle (H2O).
In some experiments, cells were incubated with variable concentrations
of BzATP or ATP, as indicated, for 5 min at 37°C. Cells were washed
with ice-cold PBS and lysed with 300 µl of 2× Laemmli sample buffer
[20 mM sodium phosphate, pH 7.0, 0.04% (wt/vol) bromphenol blue, 20%
(vol/vol) glycerin, 4% (wt/vol) SDS, and 100 mM DTT]. Samples were
sonicated for 2 s and heated for 5 min at 96-100°C,
subjected to 12% SDS-PAGE, and transferred to nitrocellulose membranes
for protein immunoblotting. Immunoblotting for phospho-ERK1/2,
phospho-MEK1/2, and phospho-PKC was performed by using a 1:1,500
dilution of mouse monoclonal anti-phospho-p44/p42 MAPK
(Thr202/Tyr204) or rabbit polyclonal
anti-phospho-MEK1/2 (Ser217/221) or a 1:1,000 dilution of
rabbit polyclonal anti-phospho-PKC (Thr505),
respectively, and then using a 1:2,000 dilution of HRP-conjugated anti-mouse or anti-rabbit IgG as the secondary antibody.
Chemiluminescence associated with specific protein bands in membranes
was visualized on autoradiographic film with the Luminol
chemiluminescence system. For normalization of the signal, the
membranes were stripped of antibodies by 30 min of incubation at 60°C
in stripping buffer [62.5 mM Tris · HCl, pH 6.8, 100 mM
2-mercaptoethanol, and 2% (wt/vol) SDS], washed in TBST [20 mM
Tris · HCl, pH 7.4, 150 mM NaCl, and 0.1% (vol/vol) Tween
20], and reprobed with a 1:2,000 dilution of rabbit anti-ERK1 (K-13)
MAPK or rabbit anti-MEK1/2 or a 1:1,000 dilution of rabbit anti-nPKC
(C-17), respectively, and a 1:2,000 dilution of HRP-conjugated
anti-rabbit IgG as the secondary antibody.
Pyk2 phosphorylation.
Human 1321N1 astrocytoma cells stably transfected with the
rP2X7-pLXSN vector were grown and serum-starved as
described in Expression of P2X7 receptors and cell
culture. Cells were then stimulated in serum-free DMEM for
5 min at 37°C, with concentrations of BzATP, as indicated. Cells were
washed with ice-cold PBS and lysed with 300 µl of Laemmli sample
buffer. Samples were sonicated for 2 s and heated for 5 min at
96-100°C, subjected to 10% SDS-PAGE, and transferred to
nitrocellulose membranes for protein immunoblotting. Immunoblotting for
Pyk2-phospho-Y402 and Pyk2-phospho-Y881 was
performed by using a 1:1,000 or 1:625 dilution of rabbit polyclonal
anti-Pyk2-p[Y402] or anti-Pyk2-p[Y881],
respectively, and a 1:2,000 dilution of HRP-conjugated anti-rabbit IgG
as the secondary antibody. Detection of specific protein bands and
normalization of the signal was performed, as described in ERK1/2, MEK1/2, and PKC phosphorylation.
Membranes were reprobed with a 1:500 dilution of rabbit anti-Pyk2, and
a 1:2,000 dilution of HRP-conjugated anti-rabbit IgG was used as the
secondary antibody.
Statistical analysis. Results are expressed as means ± SE. Data were analyzed by one-way ANOVA with Dunnett's post hoc test.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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P2X7 receptor-mediated ERK1/2 activation.
To determine whether the P2X7 nucleotide receptor can
mediate ERK1/2 activation, the recombinant rat P2X7
(rP2X7) receptor was stably expressed in human 1321N1
astrocytoma cells (1321N1/rP2X7) that lack endogenous
P2X7-R and P2Y receptors. As demonstrated by RT-PCR with
the use of specific oligonucleotide primers, cells transfected with the
rP2X7-R cDNA showed amplification products for
rP2X7-R only (Fig. 1),
whereas cells transfected with pLXSN (1321N1/pLXSN) showed no
amplification products (data not shown). BzATP, a potent
P2X7-R agonist (11, 42), increased
phosphorylation of the MAPKs ERK1/2 in 1321N1/rP2X7 but not
1321N1/pLXSN cells (Fig.
2). A 5-min
incubation with BzATP increased ERK1/2 phosphorylation in a
dose-dependent manner with a maximal response obtained with 50 µM
BzATP (Fig. 2A). The nonselective P2Y and P2X-R agonist ATP
also increased ERK phosphorylation in 1321N1/rP2X7 cells
with a maximal response induced by 3 mM ATP (Fig. 2B). BzATP
(Fig. 2C) or ATP (Fig. 2D) did not cause
significant phosphorylation of ERK1/2 in 1321N1/pLXSN cells, indicating
that ERK1/2 phosphorylation induced by BzATP or ATP in
1321N1/rP2X7 cells was mediated by the P2X7-R
and not by endogenous nucleotide receptors. Phosphorylation of ERK1/2
in 1321N1/rP2X7 cells induced by BzATP was inhibited by a
2-h treatment with 500 µM oATP (Fig.
3), an antagonist of P2X7-R
(1), confirming that rP2X7-R can couple to the
MAPK cascade.
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Phosphorylation of ERK1/2 in 1321N1 cells expressing the
rP2X7 receptor is mediated by increases in the
intracellular Ca2+ concentration.
P2X7 nucleotide receptors are ligand-gated ion channels
that permit the cellular uptake of extracellular Ca2+,
among other ions (9, 13, 37, 52, 57). To determine whether
the activation of ERK1/2 in 1321N1/rP2X7 cells was linked to an increase in the concentration of intracellular Ca2+
([Ca2+]i), we introduced the Ca2+
chelator BAPTA into cells via a 30-min pretreatment with BAPTA-AM before the addition of BzATP (Fig.
4A). Significant inhibition of
BzATP-induced ERK1/2 phosphorylation was observed at >10 µM BAPTA-AM
(P < 0.01), strongly suggesting that rP2X7
receptor-mediated ERK1/2 phosphorylation was dependent on an increase
in [Ca2+]i. Moreover, chelation of
extracellular Ca2+ by the addition of >2.5 mM EGTA to the
1.8 mM Ca2+-containing medium (Fig. 4B) also
inhibited BzATP-induced ERK1/2 phosphorylation, suggesting a role for
extracellular Ca2+ in mediating ERK1/2 phosphorylation via
rP2X7-R.
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Signal transduction pathway for ERK1/2 phosphorylation mediated by
the rat P2X7 receptor.
We examined the possibility that the rP2X7-R served to
activate Pyk2, a Ca2+-dependent, nonreceptor tyrosine
kinase that has been suggested to play a role in a variety of
cellular processes including MAPK activation (28, 31).
Activation of the rP2X7-R by BzATP caused a threefold
stimulation of Pyk2 phosphorylation on Y402 and
Y881 (Fig. 5).
Stimulation of pLXSN cell transfectants did not cause significant
phosphorylation of Pyk2 (data not shown). Treatment of the cells with
>5 µM BAPTA-AM significantly decreased phosphorylation of
Y402 and Y881 (Fig.
6), consistent with a role for
Ca2+ in Pyk2 activation. Autophosphorylation of
Y402 on Pyk2 serves to increase kinase activity and
provides docking sites for other signaling proteins, including
phosphorylated c-Src (3, 31, 50). Phosphorylation of
Y881 on Pyk2 results in additional protein-protein
interactions including binding to Grb2 and the p85 subunit of PI 3-K
and subsequent activation of the MAPK/ERK1/2 pathway (3, 31,
50). Accordingly, we found that inhibition of c-Src with >5
µM PP2 (19) (Fig.
7A) or inhibition of PI 3-K
with 
10 µM LY-294002 (56) (Fig. 7B) decreased BzATP-induced ERK1/2 phosphorylation in
1321N1/rP2X7 cells by as much as 60 or 70%, respectively,
at the highest inhibitor concentrations tested. These results indicate
that c-Src and PI 3-K are two components of the ERK1/2 signaling
pathway coupled to the rP2X7-R in 1321N1 cells. We also
detected phosphorylation of the MAPK kinase, MEK1/2, in
1321N1/rP2X7 cells incubated with BzATP (Fig.
8A), which was inhibited by a
30-min pretreatment with >0.01 µM U0126 (Fig. 8B), a
specific MEK1/2 inhibitor (12). Cells transfected with
pLXSN did not show any significant phosphorylation of MEK1/2 in the
presence of BzATP (data not shown). These results are consistent with
the positioning of MEK1/2 upstream of ERK1/2 in the MAPK cascade.
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PKC involvement in ERK1/2 phosphorylation mediated by the
P2X7 receptor.
The PKC inhibitor GF-109203X (Fig.
9A) decreased P2X7
receptor-mediated ERK1/2 phosphorylation induced by BzATP in
1321N1/rP2X7 cells. Approximately 50% inhibition
(P < 0.05) of ERK1/2 phosphorylation induced by BzATP
was observed at 5-10 µM GF-109203X, an inhibitor of
Ca2+-dependent and -independent PKC isoforms
(35). Because GF-109203X is more potent against
Ca2+-dependent than -independent PKC isoforms
(35), the relatively high level of GF-109203X needed to
inhibit ERK1/2 phosphorylation induced by BzATP suggests that a
Ca2+-independent PKC isoform may be involved. Consistent
with this conclusion, a selective inhibitor of
Ca2+-dependent isoforms of PKC, Gö-6976, only
partially inhibited BzATP-induced ERK1/2 phosphorylation at
concentrations >50 µM (Fig. 9B), concentrations that may
not reflect specific inhibition of PKC (35). Results with
these PKC inhibitors suggest that ERK1/2 phosphorylation mediated by
the rP2X7-R involves the activation of a
Ca2+-independent PKC isoform. Accordingly, stimulation of
1321N1/rP2X7 cells by BzATP induced a dose-dependent
phosphorylation of Ca2+-independent PKC on
Thr505 (Fig. 10).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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It is well known that a variety of receptors are linked to the
stimulation of ERK activity, thereby regulating cell proliferation and
differentiation (34, 38). Activation of some P2Y
nucleotide receptor subtypes has been shown to stimulate ERK
phosphorylation (10, 27, 30, 39, 40). However, not much is
known about the possible coupling of P2X-R to the MAPK cascade.
Recently, Swanson et al. (54) have reported that
P2X2 receptors in PC-12 cells could activate the MAPKs ERK1
and ERK2. More recently, the P2X7-R has been found to
activate the MAPKs SAPK/JNK in human and rodent macrophages
(21) and ERK1/2 in rat primary astrocytes (43). However, the pathway from the P2X7-R to
ERK1/2 has not been described. The present study provides direct
evidence that the P2X7-R can mediate ERK1/2 activation
through a cellular pathway that is dependent on intracellular and
extracellular Ca2+, Pyk2, c-Src, PI 3-K, and MEK1/2. We
also have determined that Ca2+-independent PKC can
couple the P2X7-R to the ERK1/2 signaling pathway. A
similar mode of signaling to ERK1/2 has been identified for G
protein-coupled P2Y2 receptors (10, 39, 51).
Our studies have shown that exposure of 1321N1 astrocytoma cells stably expressing rP2X7 to the P2X7-R agonists BzATP or ATP resulted in a dose-dependent increase in ERK1/2 phosphorylation (Fig. 2, A and B). The data indicate that this effect is due entirely to the activation of the P2X7-R, because cells transfected with the pLXSN vector alone did not show any significant ERK1/2 phosphorylation when incubated with BzATP or ATP (Fig. 2, C and D). Moreover, treatment of 1321N1/P2X7 cells with oxidized ATP, an effective P2X7-R antagonist (1), inhibited BzATP-induced ERK1/2 activation (Fig. 3). The absence of Ca2+-mobilizing P2Y and P2X receptors other than P2X7 in 1321N1/rP2X7 cells was supported by RT-PCR experiments that did not detect the presence of mRNA for P2Y1, P2Y2, P2Y4, P2Y6, P2X1, P2X2, or P2X4 receptors (Fig. 1) and by the absence of nucleotide-stimulated increases in [Ca2+]i in untransfected 1321N1 cells (15, 44).
It is well accepted that an increase in [Ca2+]i activates a wide range of intracellular responses, including in some cases MAPK activation. Possible biological mechanisms by which these increases in [Ca2+]i can occur include 1) the mobilization of intracellular Ca2+ stores and/or 2) the opening of plasma membrane channels that facilitate the influx of extracellular Ca2+. In the case of nucleotide receptors, the metabotropic P2Y receptors are known to mobilize intracellular Ca2+ from inositol 1,4,5-trisphosphate-sensitive stores (9, 58), whereas activation of ligand-gated ion channels such as P2X receptors can mediate an influx of extracellular Ca2+ and an increase in [Ca2+]i (9, 41, 42). Among the P2X receptor subtypes, it is well described that activation of the P2X7-R can lead to Ca2+ influx (9, 41, 42). An increase in [Ca2+]i following P2X7-R stimulation by BzATP apparently plays a role in ERK1/2 activation in 1321N1/P2X7 cells, because phosphorylation of ERK1/2 was inhibited by introduction of the intracellular Ca2+ chelator, BAPTA (Fig. 4A). Moreover, chelation of extracellular Ca2+ by EGTA also inhibited BzATP-induced ERK1/2 phosphorylation, suggesting that Ca2+ influx may be involved in P2X7 receptor-mediated ERK1/2 activation (Fig. 4B). In addition to Ca2+ chelation, EGTA may also decrease the pH of the medium and consequently inhibit P2X7 receptor activation that occurs with an alkaline pH optimum (11, 57). However, these results are similar to a previous study, which showed that P2X2 receptor-mediated ERK1/2 activation in PC-12 cells was dependent only on extracellular Ca2+ influx via the P2X2 receptor (54).
The MAPKs ERK1/2 are responsible for propagation of mitogenic signals
in response to growth factor stimulation, resulting in changes in
cellular morphology, metabolism, and gene expression (34,
38). However, the cellular components linking the
P2X7-R to ERK1/2 are not well understood. Our results
indicate that P2X7-R activation in 1321N1/rP2X7
cells serves to stimulate intracellular signaling molecules, including
the proline rich/Ca2+-activated tyrosine kinase Pyk2, by
inducing the phosphorylation of Pyk2 on Y402 and
Y881 (Fig. 5), which can be inhibited by introduction into
cells of the Ca2+ chelator BAPTA (Fig. 6). This
Ca2+-dependent activation of Pyk2 is consistent with
results of Lev et al. (31), who have demonstrated
that Pyk2 activation occurs upon elevation of
[Ca2+]i in PC-12 cells following stimulation
of the nicotinic acetylcholine receptor. More recently, the
P2X2 receptor has been reported to activate ERK1/2 through
Pyk2 in a Ca2+-dependent manner (54). Although
Pyk2 lacks the Src homology (SH2 and SH3) domains found in many other
soluble protein kinases, it has been shown that phosphorylation of
Y402 on Pyk2 provides docking sites for signaling proteins
such as pp60src (3, 31).
Phosphorylation of Y881 on Pyk2 provides additional sites
for protein-protein interactions, which could lead to the recruitment
of Grb2 and the p85 subunit of PI 3-K, signaling intermediates in the
ERK1/2 pathway (3). It also has been reported that
Ca2+-dependent activation of Pyk2 induces MAPK activation
via a direct interaction with c-Src (8, 36). Moreover, the
formation of a Pyk2/Src complex via Src binding to Y402 on
Pyk2 enables Src to 1) phosphorylate Pyk2 on
Y881 and within the catalytic domain (Y579 and
Y580), which promotes Grb2 binding and enhances Pyk2 kinase
activity, respectively (3), and 2)
phosphorylate adjacent cellular proteins, such as the adapter molecule
Shc (3). In addition, tyrosine phosphorylation of Shc by
Src could lead to interaction of Shc with Grb2, which serves to recruit
the guanine nucleotide exchange factor Sos, leading to Ras activation
and, ultimately, to ERK1/2 phosphorylation (2, 49). As
mentioned earlier, we have found that stimulation of the
rP2X7-R leads to an increase in the phosphorylation of
Y402 and Y881 on Pyk2, suggesting that
rP2X7 receptor-mediated ERK1/2 activation might involve the
formation of Pyk2/Src/Shc and Pyk2/Src/Grb2 complexes. In agreement
with this hypothesis, the treatment of 1321N1/rP2X7 cells
with the c-Src inhibitor PP2 inhibited rP2X7 receptor-mediated ERK1/2 activation (Fig. 7A). We also have
shown that inhibition of PI 3-K by LY-294002 decreased
rP2X7 receptor-mediated ERK1/2 phosphorylation (Fig.
7B). Similar results have been obtained with U937 monocytic
cells, where PI 3-K inhibition by LY-294002 also decreased
P2Y2 receptor-mediated ERK1/2 activation (51). PI 3-K has been shown to be involved in the activation of the ERK1/2
MAPK pathway in endothelial cells via the activation of p21ras (22). Activation of PI 3-K
has been linked to Pyk2 phosphorylation on Y881 through the
interaction of the p85 
-subunit of PI 3-K with p130CAS,
as suggested by Rocic et al. (49) in vascular smooth
muscle cells.
Using several approaches, we have also shown that ERK1/2 activation by
the rP2X7-R can occur via a Ca2+-independent
PKC. First, the Ca2+-dependent PKC inhibitor Gö-6976
(Fig. 9B) was ineffective at concentrations that inhibit PKC
as described in the literature (35), whereas GF-109203X,
at concentrations (10 µM) that inhibit both
Ca2+-dependent and -independent PKC isoforms, reduced
P2X7 receptor-mediated ERK1/2 phosphorylation in
1321N1/rP2X7 cells by ~50% (Fig. 9A). Second,
we found that phosphorylation of PKC on Thr505 was
stimulated by BzATP (Fig. 10). Previous studies with primary cultures
of rat cortical astrocytes have linked the activation of P2Y-Rs to
PKC through the hydrolysis of phosphatidylcholine by phospholipase D
(PLD) generating phosphatidic acid that can be converted to the PKC
activator diacylglycerol (DAG) by phosphatidic acid phosphohydrolase
(39). P2X7-R activation also has been shown to
activate PLD (20), which presumably could provide DAG to
activate PKC. PLD activation can be mediated by elevations in
[Ca2+]i, as reported in rat parotid acini
(17). Another possible mechanism for PKC activation
could involve inositol phosphate generation via the activity of
phosphatidylinositol 4-kinase, as recently proposed by Kim et al.
(25), which could lead to the production of lipids
required for DAG generation (25). Although PKC is
classified as a Ca2+-independent PKC isozyme, it may still
lie downstream of Ca2+-dependent Pyk2 in the
P2X7 receptor signaling pathway, because chelation of
intracellular Ca2+ (Fig. 4A) or inhibition of
Ca2+-independent PKC (Fig. 9) nearly completely inhibited
BzATP-induced ERK activation.
In summary, this study provides a detailed characterization of the
signal transduction pathway linking a P2X7 nucleotide
receptor to ERK1/2 activation. We conclude that activation of ERK1/2 by BzATP in 1321N1/P2X7 cells occurs via
Ca2+-dependent Pyk2 and couples to the activation of c-Src,
PI 3-K, and MEK1/2. In addition, our data suggest that
P2X7-R-mediated ERK activation may involve
Ca2+-independent PKC. However, the precise mechanisms of
PKC activation by the P2X7-R and the positioning of
PKC in the signaling pathway remain to be determined, but they
likely involve the generation of DAG. Thus PKC may be located
downstream of Pyk2, where it could be activated by DAG through
P2X7-mediated PLD activation (9, 20). This
coupling of the P2X7-R to the ERK signaling pathway may
play a significant role in brain disorders, because nucleotide
receptors, especially the P2X7-R, have been implicated in
astrocyte-mediated inflammation and neurodegeneration (for review, see
Ref. 47).
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants 1P01-AG-18357 and 1P90-RR-15565 and by the F21C Program of the University of Missouri-Columbia. F. P. Gendron has a postdoctoral fellowship from "Le Fonds" pour la Formation de Chercheurs et d'Aide à la Recherche du Quebec, Canada.
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FOOTNOTES |
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Present address of P. M. Theiss: Virginia Mason Research Center, Benaroya Research Institute, 1201 Ninth Ave., Seattle, WA 98101-2795.
Address for reprint requests and other correspondence: F. P. Gendron, Dept. of Biochemistry, Univ. of Missouri-Columbia, M121 Medical Sciences Bldg., Columbia, MO 65212 (E-mail: gendronf{at}health.missouri.edu or fpgendron{at}hotmail.com).
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.
10.1152/ajpcell.00286.2002
Received 21 June 2002; accepted in final form 27 September 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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) reverse transcriptase. The only amplification product
obtained was amplified with the P2X7-R primers.
