Mechanisms of P2X7 receptor-mediated ERK1/2 phosphorylation in human astrocytoma cells

Fernand-Pierre Gendron1, Joseph T. Neary2, Patty M. Theiss1, Grace Y. Sun1, Fernando A. Gonzalez3, and Gary A. Weisman1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 Cdelta 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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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 Cdelta (PKCdelta ). 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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-PKCdelta (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-nPKCdelta (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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Table 1.   Sequence-specific oligonucleotide primers used for RT-PCR studies

ERK1/2, MEK1/2, and PKCdelta 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-PKCdelta 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-PKCdelta (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-nPKCdelta (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 PKCdelta 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.


    RESULTS
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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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Fig. 1.   Determination of P2Y (A) and P2X (B) receptor mRNA expression in 1321N1/rP2X7 astrocytoma cells by RT-PCR. cDNA derived from mRNA of 1321N1 cells expressing the rat P2X7 receptor (rP2X7-R) was amplified with the indicated primers. These primers proved specific for amplification of the indicated receptor cDNA in RT-PCR with mRNA isolated from 1321N1 cell transfectants expressing the corresponding P2 receptor subtype (data not shown). Controls were run with (+) or without (-) reverse transcriptase. The only amplification product obtained was amplified with the P2X7-R primers.



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Fig. 2.   Dose-dependent activation of ERK1/2 by the recombinant rP2X7-R expressed in 1321N1 cells. Cells (1321N1/rP2X7) incubated in serum-free DMEM were stimulated for 5 min at 37°C with 0-300 µM 2',3'-O-(4-benzoyl)benzoyl-ATP (BzATP; A) or 0-5 mM ATP (B). Negative control 1321N1 cells transfected with the pLXSN vector were assayed for ERK activation after stimulation with 0-300 µM BzATP (C) or 0-5 mM ATP (D). ERK1/2 phosphorylation was detected by Western analysis (a representative blot is shown for A), and bands were quantified by densitometry. Band intensities are expressed as fold increase over the control. Results represent means ± SE of at least 3 separate experiments run in duplicate and normalized for differences in total protein. **P < 0.01.



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Fig. 3.   P2X7 receptor-mediated ERK1/2 activation is inhibited by oxidized ATP (oATP). Human 1321N1 cells expressing the recombinant rP2X7-R were incubated in serum-free DMEM, treated for 2 h at 37°C with 250 or 500 µM oATP, and then stimulated for 5 min at 37°C with 50 µM BzATP. ERK1/2 phosphorylation was detected by Western analysis (a representative blot is shown), and bands were quantified by densitometry. Band intensities are expressed as percentages of maximum values after subtraction of the control value (0 µM BzATP). Results represent means ± SE of at least 3 separate experiments run in duplicate and normalized for differences in total protein. **P < 0.01.

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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Fig. 4.   Inhibition of P2X7 receptor-mediated ERK1/2 phosphorylation by Ca2+ chelation. Human 1321N1 cells expressing the recombinant rP2X7-R were incubated in serum-free DMEM and treated with 0-50 µM BAPTA-AM for 30 min at 37°C (A) or with 0-8 mM EGTA for 5 min and then stimulated for 5 min at 37°C with 50 µM BzATP (B). ERK1/2 phosphorylation was detected by Western analysis, and bands were quantified by densitometry. Band intensities are expressed as percentages of maximum values after subtraction of the control value (0 µM BzATP). Results represent means ± SE of at least 3 separate experiments run in duplicate and normalized for differences in total protein. **P < 0.01.

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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Fig. 5.   Pyk2 phosphorylation on tyrosines 402 (Y402) and 881 (Y881) in 1321N1/rP2X7 cells induced by BzATP. Cells were incubated in serum-free DMEM for 5 min at 37°C with the indicated concentration of BzATP (0-300 µM), site-specific phosphorylation of Pyk2 on Y402 (A) or Y881 (B) was detected by Western analysis (representative blots are shown), and bands were quantified by densitometry. Band intensities are expressed as fold increase over the control (0 µM BzATP). Results represent means ± SE of at least 3 separate experiments run in duplicate and normalized for differences in total protein. *P < 0.05.



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Fig. 6.   Pyk2 phosphorylation is inhibited by intracellular Ca2+ chelation in 1321N1/rP2X7 cells. Cells were incubated in serum-free DMEM for 30 min at 37°C with 0-50 µM BAPTA-AM and then stimulated for 5 min at 37°C with 50 µM BzATP. A: Pyk2 phosphorylation on Y402 or Y881 was detected by Western analysis. B: bands were quantified by densitometry. Band intensities are expressed as a percentage of the control (0 µM BzATP). Results represent means ± SE of at least 3 separate experiments run in duplicate and normalized for differences in total protein, where filled bars represent Pyk2-p[Y402] and open bars represent Pyk2-p[Y881]. Statistical significance (**P < 0.01) was determined using a one-way ANOVA test.



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Fig. 7.   Inhibition of P2X7 receptor-mediated ERK1/2 phosphorylation by inhibitors of c-Src and phosphatidylinositol 3-kinase (PI 3-K). Human 1321N1/rP2X7 cells were incubated in serum-free DMEM for 30 min at 37°C with 0-50 µM PP2 (c-Src inhibitor; A) or 0-100 µM LY-294002 (PI 3-K inhibitor; B) and then stimulated with 50 µM BzATP for 5 min at 37°C. ERK1/2 phosphorylation was detected by Western analysis, and bands were quantified by densitometry. Band intensities are expressed as percentages of maximum values after subtraction of the control value (0 µM BzATP). Results represent means ± SE of at least 3 separate experiments run in duplicate and normalized for differences in total protein. *P < 0.05; **P < 0.01.



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Fig. 8.   Phosphorylation of MEK1/2 in 1321N1/rP2X7 cells stimulated by BzATP. Cells were incubated in serum-free DMEM with 0-300 µM BzATP for 5 min at 37°C (A) or with 0-10 µM U0126 for 30 min at 37°C (B) and then stimulated with 50 µM BzATP for 5 min at 37°C. ERK1/2 phosphorylation was determined by Western analysis (a representative blot is shown in A), and bands were quantified by densitometry. Band intensities are expressed as either fold increase over control (0 µM BzATP) in A or percentages of maximum values after subtraction of the control value (0 µM BzATP) in B. Results represent means ±SE of at least 3 separate experiments run in duplicate and normalized for differences in total protein. **P < 0.01.

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 PKCdelta on Thr505 (Fig. 10).


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Fig. 9.   The effects of PKC inhibition on P2X7 receptor-mediated ERK1/2 phosphorylation. Human 1321N1/rP2X7 cells were incubated in serum-free DMEM for 30 min at 37°C with the indicated concentrations of the PKC inhibitors GF-109203X (A) or Gö-6976 (B) and then stimulated for 5 min at 37°C with 50 µM BzATP. ERK1/2 phosphorylation was detected by Western analysis, and bands were quantified by densitometry. Band intensities are expressed as percentages of maximum values after subtraction of the control value (0 µM BzATP). Results represent means ±SE of at least 3 separate experiments run in duplicate and normalized for differences in total protein. *P < 0.05; **P < 0.01.



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Fig. 10.   P2X7 receptor-mediated PKCdelta phosphorylation on Thr505. Human 1321N1/rP2X7 cells were incubated in serum-free DMEM for 5 min at 37°C with 0-300 µM BzATP, PKCdelta phosphorylation on Thr505 was determined by Western analysis (a representative blot is shown), and bands were quantified by densitometry. Band intensities are expressed as fold increase over control (0 µM BzATP). Results represent means ± SE of at least 3 separate experiments run in duplicate and normalized for differences in total protein. **P < 0.01.


    DISCUSSION
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INTRODUCTION
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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 PKCdelta 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 alpha -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 PKCdelta 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 PKCdelta 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 PKCdelta . PLD activation can be mediated by elevations in [Ca2+]i, as reported in rat parotid acini (17). Another possible mechanism for PKCdelta 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 PKCdelta 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 PKCdelta . However, the precise mechanisms of PKCdelta activation by the P2X7-R and the positioning of PKCdelta in the signaling pathway remain to be determined, but they likely involve the generation of DAG. Thus PKCdelta 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).


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 284(2):C571-C581