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J. Biol. Chem., Vol. 275, Issue 35, 26792-26798, September 1, 2000

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Stress-activated Protein Kinase/JNK Activation and Apoptotic Induction by the Macrophage P2X7 Nucleotide Receptor^*

Benjamin D. Humphreys, Janet Rice, Sylvia B. Kertesy, and George R. Dubyak^§

From the Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106

Received for publication, April 2, 2000, and in revised form, June 13, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In human and rodent macrophages, activation of the P2X7 nucleotide receptor stimulates interleukin-1 processing and release, apoptosis, and killing of intracellular Mycobacterium tuberculosis. Signaling pathways downstream of this ionotropic ATP receptor are poorly understood. Here we describe the rapid activation of the stress-activated protein kinase (SAPK)/JNK pathway in BAC1 murine macrophages stimulated by extracellular ATP. Brief exposure of the cells to ATP (10-30 min) was sufficient to trigger a rapid accumulation of activated SAPK that was then sustained for >120 min. Several observations indicated that the P2X7 receptor mediated this effect. 1) ATP and 3'-O-(4-benzoyl)benzoyl-ATP were the only agonistic nucleotides. 2) The effect was inhibited by oxidized ATP and the isoquinoline KN-62, two known P2X7 receptor antagonists. 3) ATP-induced SAPK activation could be recapitulated in P2X7 receptor-transfected HEK293 cells, but not in wild-type HEK293 cells. Because P2X7 receptor stimulation can rapidly activate caspase family proteases that have been implicated in the induction of the SAPK pathway, we investigated whether ATP-dependent SAPK activation involved such proteases. Brief exposure of BAC1 macrophages to extracellular ATP induced DNA fragmentation, -fodrin breakdown, and elevated levels of caspase-3-type activity. Asp-Glu-Val-Asp-cho, a caspase-3 inhibitor, inhibited ATP-induced DNA fragmentation and -fodrin proteolysis, but had no effect on ATP-induced SAPK activation. Tyr-Val-Ala-Asp-chloromethyl ketone, a caspase-1 inhibitor, prevented ATP-induced release of processed interleukin-1, but not ATP-dependent SAPK activity. We conclude that activation of ionotropic P2X7 nucleotide receptors triggers a strong activation of SAPK via a pathway independent of caspase-1- or caspase-3-like proteases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The P2X7 nucleotide receptor belongs to the P2X family of ATP-gated ion channels. This family comprises seven distinct gene products, each possessing two putative membrane-spanning domains with intracellular N and C termini (for review, see Refs. 1 and 2). The P2X7 receptor is identical to the functionally defined P2Z receptor and is expressed primarily in hematopoietic cells and a limited number of other cell types including parotid acinar cells, testis, and fibroblasts (3-5). As a non-desensitizing, nonselective cation channel with low affinity for ATP, P2X7 receptor activation requires millimolar extracellular ATP in the presence of divalent cations. Channel opening triggers rapid depolarization, calcium influx, and equilibration of sodium and potassium gradients. Because individual P2X7 receptor proteins contain only two transmembrane-spanning segments, it is assumed that the functional channels are oligomeric complexes composed of several individual subunits. Recent studies indicate that recombinant P2X7 receptor subunits can self-assemble during in vitro translation and processing into stable, detergent-resistant complexes (6). P2X7 receptor activation additionally induces a nonselective pore able to pass molecules up to 800 Da, a characteristic shared to a lesser degree by other P2X members (7, 8). Pore structure remains uncharacterized. The ability of heterologously expressed P2X7 receptors to reconstitute ATP-dependent channel/pore formation has been interpreted as evidence that the pore reflects either further multimerization of the P2X7 channels or a dynamic change in the selectivity filter of P2X7 channels (9). Other data suggest that the channel and the pore are separate entities (10).

Depending on cell background, activation of the P2X7 receptor triggers diverse physiologic processes. For example, human monocytes primed by bacterial endotoxin/lipopolysaccharide (LPS)¹ respond to extracellular ATP with the caspase-1-dependent proteolytic maturation and externalization of IL-1 (11-13). Apoptosis is another prominent consequence of P2X7 receptor activation in various types of leukocytes. Zanovello et al. (14) first demonstrated ATP-dependent apoptosis in a lymphocyte cell line, and Zheng et al. (15) confirmed this observation in murine thymocytes. Hogquist et al. (11) later showed that brief (30 min) exposure of thioglycolate-elicited murine peritoneal macrophages to ATP was sufficient to initiate the signaling cascade that leads to apoptotic death. Subsequent studies have specifically implicated the P2X7 receptor in mediating ATP-induced apoptosis of human macrophages, mesangial cells, dendritic cells, and microglial cells (16-19). As with most examples of apoptosis, the P2X7 receptor-initiated cascade involves a defined sequence of phenotypic changes that culminate in death only several hours after the transient exposure to ATP. Agonist-occupied P2X7 receptors also drive signals that induce nuclear accumulation of various activated transcription factors, such as NFAT within minutes or NF-B within hours (20, 21). The P2X7 receptor-induced proteolytic processing and release of IL-1 from LPS-primed macrophages also precede cell death (13). These observations suggest that in the time period between commitment to apoptosis and actual cell death, P2X7 receptor activation triggers additional signals, such as NFAT activation or IL-1 release, that modulate the overall inflammatory response of macrophages. This possibility is supported by the observation that brief ATP pulses trigger not only macrophage apoptosis, but also killing of intracellular mycobacteria, including virulent Mycobacteria tuberculosis (22). Because other inducers of macrophage apoptosis do not kill M. tuberculosis, an as yet unidentified P2X7-specific signal presumably induces killing of internalized M. tuberculosis before the macrophage itself dies (16, 23). The biochemical steps linking channel/pore activation to IL-1 release, accumulation of pro-inflammatory transcription factors, M. tuberculosis killing, and macrophage apoptosis are poorly understood.

The stress-activated protein kinases (SAPKs; also known as JNKs) phosphorylate and activate transcription factors such as ATF2, Ets, and c-Jun in response to diverse cell stressors. These include UV radiation, osmotic shock, inflammatory cytokines, and endoplasmic reticulum stress (for review, see Refs. 24 and 25). Many of the downstream effectors of SAPK signaling contribute to the inflammatory response, including the TNF--dependent induction of E-selectin, NF-B induction in T cells, and both pro-apoptotic and anti-apoptotic effects in a variety of cell types (24). 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 expression of both the Jnk1 and Jnk2 genes (55).

Proximally, SAPKs are activated by the dual-specificity mitogen-activated protein kinase kinases (MEKs), which in turn are activated by the MEK kinases (MEKKs). Upstream regulators of MEKK are incompletely characterized. TNF--dependent SAPK activation is best described and involves recruitment of the adaptor protein TRAF2 to the cytosolic portion of the ligated TNF- receptor. TRAF2 mediates the activation of a series of downstream kinases that lead to phosphorylation of SAPK itself (26). Other intermediates have been proposed to play a role in different models of SAPK activation, including oxidative stress, DNA damage, and caspase proteases. Altered ion fluxes have also been associated with SAPK activation in several systems. Kuroki et al. (29) described activation of SAPK by palytoxin, a natural marine toxin from Palythoa tuberculosa that acts as a skin tumor promoter and modulator of the Na⁺,K⁺-ATPase. Moreover, palytoxin-induced SAPK activity requires sodium flux (30). Similarly, UV irradiation of myeloblastic leukemia cells induces a prominent K⁺ channel activation that, in turn, induces SAPK-dependent apoptosis (31). Since a major consequence of P2X7 receptor activation is bulk movement of both sodium and potassium, we hypothesized that P2X7 receptor activation would activate SAPK. We found that exposure of murine macrophages to short pulses of extracellular ATP can rapidly induce a sustained activation of SAPK. Pharmacological selectivity and molecular evidence indicated that the P2X7 receptor mediates this ATP-induced kinase activity. The P2X7 receptor can activate caspases involved in either cytokine processing or apoptotic induction (18, 19). Thus, we also evaluated the possible involvement of caspase-1 and caspase-3 in this P2X7 receptor-induced pathway of SAPK activation. The results indicate that although both caspase-1 and caspase-3 were activated by the pulsed ATP protocol, neither protease plays a role in coupling the P2X7 receptor to the SAPK signaling cascade.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- All nucleotides were from Sigma, except for 2'-methylthio-ATP, which was from Research Biochemicals Inc. (Natick, MA). Anisomycin and ouabain was from Sigma. The GST-Jun-(1-79) plasmid was from Dr. J. Woodgett (Woods Hole Biological Laboratory). [-³²P]ATP was from NEN Life Science Products. Anti-SAPK and anti-phospho-specific SAPK antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The anti--fodrin antibody was from Chemicon International, Inc. KN-62 (1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine) and the caspase-1 fluorogenic substrate peptide were from BIOMOL Research Labs Inc. (Plymouth Meeting, PA). The capture and detecting antibodies used for the murine IL-1 ELISA were from Endogen, Inc. (Woburn, MA). DEVD-cho and YVAD-cmk were from Bachem California. The caspase-3 fluorogenic substrate peptide was from Calbiochem. Recombinant murine TNF- and a neutralizing antibody against murine TNF- were from R&D Systems, Inc.

Cell Culture-- The BAC1.2F5 macrophage cell line, a clone of the SV40-transformed murine macrophage cell line BAC1, was maintained using previously described protocols (32). Wild-type HEK293 cells and HEK cells stably transfected with the human P2X7 receptor were maintained as described previously (33).

SAPK Assay-- JNK activation was measured according to Kuroki et al. (29, 30) with minor modifications. BAC1 cells (3 × 10⁶/ml) were plated in six-well dishes. Cultures were incubated with test agents dissolved in Iscove's Dulbecco's modified Eagle's medium supplemented with 0.1% bovine serum albumin at 37 °C. Samples were then washed once with cold phosphate-buffered saline and lysed with lysis buffer (25 mM HEPES (pH 7.7), 300 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA (pH 8.0), 0.1% Triton X-100, 0.5 mM DTT, 20 mM -glycerophosphate, 0.1 mM Na₃VO₄, 2 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride). Whole cell lysates were rotated for 30 min at 4 °C, followed by centrifugation at 10,000 × g for 10 min. Supernatant protein concentration was determined by the Bradford assay (57). 50 µg of lysate was diluted to contain 20 mM HEPES (pH 7.7), 75 mM NaCl, 2.5 mM MgCl₂, 0.1 mM EDTA, 0.05% Triton X-100, 0.5 mM DTT, 20 mM -glycerophosphate, 0.1 mM Na₃VO₄, 2 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride and mixed with 10 µl of GSH-agarose beads (Sigma) bound to the GST-Jun fusion protein. The mixture was rotated for 2 h at 4 °C, and the beads were then washed three times with HEPES binding buffer (20 mM HEPES (pH 7.7), 50 mM NaCl, 2.5 mM MgCl₂, 0.1 mM EDTA, 0.05% Triton X-100, 0.1 mM Na₃VO₄, 2 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride). Beads were resuspended in 40 µl of kinase buffer (20 mM HEPES (pH 7.6), 20 mM MgCl₂, 20 mM -glycerophosphate, 20 mM p-nitrophenyl phosphate, 0.1 mM Na₃VO₄, 2 mM DTT, 20 µM ATP, and 5 µCi of [-³²P]ATP) and incubated for 15 min at 25 °C. Proteins were eluted in SDS buffer, boiled for 3 min, and separated by SDS-polyacrylamide gel electrophoresis (12%). After Coomassie Blue staining, the gel was dried and exposed to Eastman Kodak x-ray film. GST-Jun phosphorylation was quantitated with a Bio-Rad PhosphorImager.

Western Blots-- Supernatant lysates were separated by SDS-polyacrylamide gel electrophoresis (12%) and electrophoretically transferred to polyvinylidene difluoride membranes for 15 h at 30 mV. Polyvinylidene difluoride membranes were rinsed in immunoblot buffer (10 mmol/liter Tris (pH 7.4), 0.9% NaCl, 0.05% Tween 20, and 1 mmol/liter EDTA) and blocked with milk buffer (4% nonfat dried milk (Sigma) in immunoblot buffer). After washing (1 × 15 min, 2 × 5 min) with immunoblot buffer, the polyvinylidene difluoride membranes were incubated for 1 h at room temperature with primary antibodies dissolved in milk buffer. Both anti-JNK antiserum and anti-phospho-specific JNK monoclonal antibody were used at 1 µg/ml. Membranes were then washed and incubated for 1 h with 1:5000 dilutions of horseradish peroxidase-conjugated donkey anti-rabbit (for anti-JNK antiserum) or anti-mouse (for anti-phospho-specific JNK antibody) antibody (Amersham Pharmacia Biotech). Membranes were washed and developed using chemiluminescent reagents (SuperSignal from Pierce) for 0.5-5 min and exposed to Eastman Kodak x-ray film.

Measurement of DNA Fragmentation-- At appropriate time points, 1 × 10⁶ cells (both floating and adherent) were centrifuged at 1500 × g for 30 s, resuspended in 0.5 ml of lysis buffer (5 mM Tris-HCl, 20 mM EDTA, and 0.5% Triton X-100 (pH 8.0)), and placed on ice for 15 min. Samples were then centrifuged at 12,000 × g for 20 min, and the supernatant containing DNA cleavage products was precipitated overnight using isopropyl alcohol. Pellets were resuspended in Tris/EDTA and digested with 0.2 mg/ml proteinase K and 1 mg/ml RNase A for 30 min at 37 °C. DNA fragments were separated on a 1.5% agarose gel, visualized with ethidium bromide, and photographed.

Caspase-3-like Activity-- After challenge with appropriate agonists, cell monolayers were washed once with 150 mM NaCl, 20 mM Tris-HCl, and 1 mM EDTA (pH 7.5). Cells were centrifuged for 30 s at 1500 × g, and pellets were resuspended in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 5 mM EDTA, 5 mM EGTA, and 1% Triton X-100 (pH 7.5)), incubated for 15 min at 37 °C, and centrifuged at 12,000 × g for 20 min. The supernatants were then stored at 80 °C for future use. To assess supernatant caspase-3-like activity, 0.3 ml of lysate was combined with buffer (100 mM NaHEPES, 10% glycerol, 1 mM EDTA, and 5 mM DTT (pH 7.5)) and fluorogenic caspase-3 substrate to a final concentration of 14 µM. Fluorescence was measured at an excitation wavelength of 380 nm and an excitation wavelength of 460 nm at 10-min intervals using a fluorescence spectrophotometer (Perkin-Elmer). Data were plotted, and slopes were calculated along the linear portion of the curve (at least five separate measurements). Data are presented as the slope in arbitrary units.

IL-1 ELISA-- Macrophages were plated in 12-well dishes at 5 × 10⁵/ml 1 day prior to challenge. On the day of the experiment, cells were stimulated with LPS (1 µg/ml) for 4 h to induce expression of pro-IL-1. In certain groups, the YVAD-cmk inhibitor was added for the final 30 min of LPS incubation. In the continued presence of serum-containing Dulbecco's modified Eagle's medium and LPS, cells were challenged with 3 mM ATP with or without the YVAD-cmk inhibitor. After 60 min, 5-µl aliquots of the tissue culture supernatant were assayed for IL-1 release by a sandwich ELISA. Briefly, a 96-well plate was coated with 1 µg/ml primary anti-murine IL-1 antibody overnight and then blocked with 4% bovine serum albumin in phosphate-buffered saline for 1 h. Plates were washed three times with wash buffer (50 mM Tris-HCl (pH 7.5) and 0.2% Tween 20). 5-µl aliquots of tissue culture media or murine IL-1 standards, diluted with 45 µl of Dulbecco's modified Eagle's medium, were added to the blocked wells together with 50 µl of a second, biotinylated anti-murine IL-1 antibody (at 0.2 µg/ml). The plates were incubated at room temperature for 2 h and then washed three times. The captured immune complexes were colorimetrically detected by subsequent incubations with streptavidin-horseradish peroxidase conjugate and tetramethylbenzidine substrate for horseradish peroxidase.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

SAPK Is Activated by Stimulation with Extracellular ATP-- One of the early effects of P2X7 receptor activation is equilibration of the trans-plasma membrane K⁺ and Na⁺ gradients. Since ionic perturbation can trigger SAPK signaling in certain cell types, we investigated the ability of P2X7 receptors to couple to SAPK. As an experimental approach, BAC1 murine macrophages were pulse-stimulated with extracellular ATP for relatively brief periods ranging from 10 to 30 min. Brief ATP pulses may be a more "physiologic" stimulus since tissue macrophages are likely to be exposed to extracellular ATP for only short times due to the widespread expression of ectoapyrases, which enzymatically degrade extracellular ATP. Although removal of extracellular divalent cations can increase P2X7 receptor affinity for ATP, tissue macrophages are continuously exposed to millimolar levels of extracellular Mg²⁺ and Ca²⁺. For this reason, tissue culture medium (Iscove's Dulbecco's modified Eagle's medium) containing normal levels of divalent cations was employed in all experiments. In the experiment illustrated in Fig. 1, BAC1 cells were pulsed with 5 mM ATP. After a 30-min incubation, the ATP-containing medium was aspirated and replaced with normal ATP-free Iscove's medium. At the indicated time points (Fig. 1, A and B), cell lysates were assessed for SAPK activity. SAPK activity was low during or immediately following the ATP pulse, but increased rapidly after ATP removal to a maximum of 4.6-fold over basal levels. The kinase remained activated even 2 h after ATP washout. Since continuous ATP exposure can trigger colloido-osmotic lysis in these cells, control experiments verified that the 30-min ATP pulse itself did not cause necrotic cell death. The cytosolic enzyme lactate dehydrogenase was used as a marker for lysis. No increase in lactate dehydrogenase was observed in the extracellular medium at any point during ATP pulses (data not shown). Cells were harvested in subsequent experiments at 60 min following the ATP pulse since the induced SAPK activity was maximal at this time. To further characterize the SAPK response, BAC1 cultures were challenged with ATP for different times (0-30 min) prior to washout. Fig. 2 shows that the magnitude of SAPK activation (measured at 60 min post-ATP removal) was dependent on the duration of the ATP pulse. A 5-min pulse was sufficient to elicit a measurable accumulation of active SAPK, and near-maximal activation was observed within 20 min.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Treatment of murine macrophages with short pulses of extracellular ATP induces sustained activation of SAPK. A, BAC1 macrophages were challenged with vehicle or with 5 mM ATP for 30 min at 37 °C. The culture medium was removed; the cells were then washed once; and fresh medium was added. Cells were incubated at 37 °C for the indicated times and lysed, and SAPK activity was assessed as described under "Experimental Procedures." B, -fold SAPK activation is plotted as a function of time for the experiment shown in A. The solid bar from 0 to 30 min indicates the vehicle or ATP pulse.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Relationship between ATP pulse duration and induction of sustained SAPK activity. A, BAC1 macrophages were challenged with vehicle or with 5 mM ATP for the indicated times; the cells were then washed once; and fresh medium was added. After a 60-min incubation at 37 °C, cells were lysed, and SAPK activity was assessed as described under "Experimental Procedures." B, -fold SAPK activation is plotted as a function of time for the experiment shown in A.

The P2X7 Receptor Mediates SAPK Activation-- BAC1 macrophages express calcium-mobilizing P2Y receptors in addition to P2Z/P2X7 receptors (34); and given the high concentration of ATP used to activate SAPK, we next determined whether the ATP-induced SAPK activity was mediated by the P2X7 receptor or by another P2 receptor. Fig. 3A shows that preincubation of macrophages with periodate-oxidized ATP blocked ATP-induced SAPK activity. In contrast, there was no inhibition of anisomycin-induced SAPK activity, indicating that the SAPK signaling pathway remained intact after oxidized ATP pretreatment. Western blot analysis using an anti-SAPK antibody verified that the assay conditions did not alter overall SAPK protein levels. Tyr and Thr phosphorylation of SAPK is required for activity, and we verified that enzymatic kinase activity increased in parallel with accumulation of phospho-SAPK immunoreactivity (Fig. 3A). Although oxidized ATP inhibits the P2X7 receptor, its specificity for the P2X7 receptor over other P2X receptors is unclear. Therefore, we verified that the isoquinoline derivative KN-62, a compound that also inhibits the P2X7 receptor in BAC1 macrophages (33), completely blocked ATP-induced SAPK activity (Fig. 3B). These pharmacological data implicate the P2X7 receptor as the P2 receptor responsible for SAPK activation.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Activation of SAPK by extracellular ATP in macrophages is mediated by the P2X7 receptor. A, BAC1 cells were incubated at 37 °C in the absence (control) or presence of 5 mM ATP or 200 nM anisomycin for 30 min. Cells were then washed once, and fresh medium lacking agonists (second control and ATP) or containing 200 nM anisomycin (Aniso) was replaced. Cells were incubated at 37 °C for 60 min before lysis. Some cells were preincubated with the P2X7 receptor antagonist oxidized ATP (+oATP; 300 µM) for 3 h prior to stimulation. These groups were then challenged with ATP or anisomycin for 30 min in the continued presence of oxidized ATP. The oxidized ATP was not replaced after the washing step. -Fold activation of SAPK was determined after quantitative PhosphorImager analysis of 32P incorporated into GST-Jun and normalized to the control lane. 30 µg of lysate from the same experiment was resolved by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting using either an anti-JNK polyclonal antiserum ( panSAPK) or an anti-phospho-specific SAPK antibody ( P-SAPK). B, BAC1 cells were incubated at 37 °C in the absence (control) or presence of 3 mM ATP or 200 nM anisomycin (Aniso) for 30 min. 5 µM KN-62 was added to some groups (+KN-62) along with the ATP or anisomycin. After 30 min, the cells were washed once, and fresh medium lacking agonists (second control and ATP) or containing 200 nM anisomycin (second Aniso) was replaced in the continued presence of KN-62. After a 60-min incubation, cells were lysed, and SAPK activity was assessed and presented as described for A. C, the indicated nucleotides were added to BAC1 cultures for 30 min at 37 °C. Cells were then washed once, and fresh medium lacking nucleotides was replaced. After the subsequent 60-min incubation, cells were lysed, and SAPK activity was determined as described for A. BzATP, 3'-O-(4-benzoyl)benzoyl-ATP; ADO, adenosine; 2-meS-ATP, 2'-methylthio-ATP.

The ability of nucleotides other than ATP to activate SAPK was also tested. Fig. 3C shows that only 3'-O-(4-benzoyl)benzoyl-ATP and ATP were SAPK agonists. Significantly, the P2Y2 agonist UTP was without effect, ruling out a contribution of these metabotropic P2 receptors expressed in BAC1 macrophages. The millimolar concentrations of ATP used to stimulate the P2X7 receptor cause strong chelation of extracellular divalent cations, and this could be contribute to the observed activation of SAPK. Since UTP also chelates divalent cations, but does not activate P2X7 receptors, the failure of 5 mM UTP to simulate any SAPK activity suggests that simple chelation of extracellular divalent cations cannot explain ATP-dependent SAPK activity (Fig. 3C). The P2Y1 agonists 2'-methylthio-ATP and ADP and the P1 agonist adenosine were also ineffective at inducing SAPK activity. This nucleotide selectivity profile is consistent with the pharmacology of both cloned and natively expressed P2X7 receptors.

Macrophages can release various inflammatory cytokines, such as IL-1 and TNF-, which also activate SAPK signaling cascades; and these cytokines can act as autocrine modulators of LPS-initiated macrophage activation (56). The delayed, but sustained, activation of SAPK that followed ATP pulsing suggested an indirect mechanism that could involve the release and gradual extracellular accumulation of TNF- and/or IL-1. However, the IL-1 and TNF- genes are transcriptionally silent in quiescent macrophages in the absence of primary inflammatory stimuli such as LPS and interferon- (56). It should be stressed that P2X7 receptor-dependent activation of SAPK was observed in BAC1 macrophages even in the absence of priming by LPS or interferon- (Figs. 1-3). Western blot analyses confirmed the absence of IL-1 or TNF- protein expression in either control BAC1 macrophages or cells stimulated with ATP pulses in the absence of LPS pretreatment (data not shown); this contrasted with the marked accumulation of these cytokines in LPS-treated BAC1 cells. The delayed accumulation of phosphorylated SAPK triggered by ATP was not attenuated when BAC1 macrophages were incubated with neutralizing anti-TNF- antibodies during and following ATP pulse stimulation (data not shown). Finally, as demonstrated in subsequent experiments (see Fig. 6 and Table I), treatment of LPS-primed BAC1 cells with an inhibitor of interleukin-converting enzyme/caspase-1 failed to attenuate ATP-induced SAPK activation despite strong inhibition of the ATP-induced IL-1 secretion. These studies indicate that release of inflammatory cytokines is an unlikely mechanism for induction of the SAPK signaling cascade by P2X7 receptors.

View this table: [in this window] [in a new window]   Table I Inhibition of IL-1 release by YVAD-cmk BAC1 macrophages were treated with LPS (1 µg/ml) for 4 h prior to challenge with 3 mM ATP for 60 min at 37 °C. Where indicated, YVAD-cmk (200 µM) was added 30 min prior to ATP challenge. 5 µl of cell supernatants was then assayed for mature IL-1 by ELISA as described under "Experimental Procedures." Values are means ± S.D.

Heterologous Expression of the P2X7 Receptor Confers ATP-sensitive SAPK Activity-- To further investigate the relationship between P2X7 receptor activation and the stimulation of SAPK, we compared the abilities of ATP to activate SAPK in wild-type HEK293 cells and in an HEK line that stably expresses the human P2X7 receptor (HEK-P2X7). To prevent detachment of cells during ATP stimulation, HEK cells were plated in poly-L-lysine-coated tissue culture dishes. Fig. 4A demonstrates that P2X7 receptor-transfected cells, but not the untransfected cells (HEK-wt), were characterized by a sustained elevation of SAPK activity following a 20-min pulse with 5 mM ATP. As a positive control, anisomycin stimulated SAPK equally well in both cell types. The accumulation of phosphorylated SAPK in HEK-P2X7 cells following the 20-min ATP pulse was characterized by a time course (Fig. 4B) that was similar to the time course observed in the BAC1 macrophages (Fig. 1B), with maximal accumulation occurring ~60 min following the removal of ATP. These experiments provide definitive evidence that the P2X7 receptor activation is sufficient for ATP induction of SAPK activity and suggest that the signaling pathway coupling P2X7 receptors to SAPK is not restricted to macrophages. Both p46 and p55 forms of SAPK in the HEK cells were similarly phosphorylated in response to P2X7 receptor activation (Fig. 4B). This contrasts with the predominant accumulation of phosphorylated p46 isoforms in the ATP-pulsed BAC1 cells (Fig. 3A). A preferential activation of p46^SAPK in mouse macrophages stimulated with TNF- has also been reported (49).

View larger version (38K): [in this window] [in a new window]   Fig. 4.   ATP-induced SAPK activity in wild-type versus P2X7 receptor-transfected HEK293 cells. A, wild-type HEK293 (HEK-wt) and HEK-P2X7 cells were plated in six-well dishes coated with poly-L-lysine at 3 × 105/ml 1 day before the experiment. Cells were challenged with 5 mM ATP for 20 min and then washed and incubated at 37 °C for 1 h and processed. Alternatively, cells were challenged with 10 µM anisomycin (Aniso) for 20 min and then processed. SAPK activity was determined as described in the legend to Fig. 1. B, HEK-P2X7 cells in six-well dishes were challenged with 5 mM ATP for 20 min and then washed and incubated in fresh medium for an additional 0, 30, 60, or 90 min prior to processing. Parallel wells were untreated or treated with 10 µM anisomycin for 60 min. 40-µg aliquots of lysate protein were resolved by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting using either an anti-JNK polyclonal antiserum (a-pan SAPK) or an anti-phospho-specific SAPK antibody (a-phospho SAPK). con, control.

Dissociation of SAPK Activation from Increased Caspase Activity-- Caspases are ubiquitously expressed cysteine proteases involved in the activation and execution of apoptosis (35). SAPK can be activated by the caspase-dependent cleavage of upstream signaling molecules such as MEKK and p21-activated protein kinase (PAK) (36, 37). Since P2X7 receptor stimulation has been associated with activation of caspase-1 and caspase-3 in other inflammatory cell types (12, 18, 19), our findings suggested two possibilities: 1) ATP-dependent SAPK activity is the secondary consequence of caspase activation, or 2) SAPK activity is upstream of caspase activation. We first verified that P2X7 receptor activation induced both caspase-3 and caspase-1 activities in BAC1 macrophages. The 30-min ATP pulse induced time-dependent oligonucleosomal DNA laddering (Fig. 5A). Fig. 5B shows that DNA laddering was preceded by the breakdown of a cytoskeletal protein, -fodrin, which is a known substrate for caspase-3 during apoptosis (38, 39). Caspase-3 activity within cell lysates (measured by the hydrolysis of a fluorogenic substrate peptide) also increased as a function of time following the ATP pulse (Fig. 5C). The increase in caspase-3 activity at 3 h closely correlated with the time course for -fodrin breakdown, observed at 4 h after the ATP pulse, supporting the notion that caspase-3 mediates -fodrin proteolysis in these cells. The amount of caspase-3 activity in cell lysates also depended on the length of the ATP pulse (Fig. 5D) in a manner similar to that which characterized the accumulation of active SAPK within lysates (Fig. 2, A and B). The caspase-3 inhibitor DEVD-cho attenuated DNA laddering in ATP-stimulated cells (Fig. 5C) and inhibited -fodrin breakdown (Fig. 5D), supporting a role for caspase-3 in these events.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Treatment of murine macrophages with short pulses of extracellular ATP induces sustained activation of caspase-3 and apoptotic induction. A, BAC1 macrophages were pulsed with 5 mM ATP for 30 min, washed, and incubated at 37 °C for the indicated times. DNA cleavage products were isolated as described under "Experimental Procedures." B, after a 30-min, 5 mM ATP pulse, cells were incubated at 37 °C for the indicated times. The ~240-kDa cytoskeletal protein -fodrin was measured using Western blot analysis of cell lysates as described under "Experimental Procedures." The two major cleavage products at ~150 and ~120 kDa result from caspase-3-dependent proteolysis (38, 39). C and D, in vitro caspase-3-like protease activity was measured from cell lysates using a fluorogenic caspase-3 substrate peptide. Cytosolic lysates were made at the indicated times after a 5 mM ATP pulse of either 30 min (C) or various times (D). E, the caspase-3-like inhibitor DEVD-cho (300 µM) was added to cultures 30 min prior to a 30-min, 5 mM ATP pulse. DEVD-cho was added back to cell cultures after washout, and DNA fragmentation was assessed 6 h after the ATP pulse. F, -fodrin breakdown was assessed 8 h after a 30-min, 5 mM ATP pulse in the presence or absence of DEVD-cho (300 µM). con, control.

We also verified that activation of the P2X7 receptor could stimulate the rapid processing of pro-IL-1 to mature IL-1 in our system. In LPS-primed macrophages, an ATP stimulus triggered the release of processed IL-1, as measured by ELISA-specific mature IL-1. Similar to previous reports, this process was mediated by the interleukin-converting enzyme/caspase-1 because the caspase-1-like inhibitor YVAD-cmk strongly attenuated the ATP-stimulated release of immunoreactive IL-1 (Table I).

P2X7 Receptor-dependent SAPK Activity Does Not Require Caspase-1- or Caspase-3-like Activity-- Having established that ATP pulses can induce both caspase-3 and caspase-1 activities in these BAC1 macrophages, we next assessed whether ATP-induced SAPK activity was caspase-dependent. Fig. 6 shows that at concentrations sufficient to inhibit their respective proteases, neither DEVD-cho nor YVAD-cmk had any effect on ATP-induced SAPK activity. These results indicate that the P2Z/P2X7 receptor couples to SAPK by a caspase-3- and caspase-1-independent mechanism.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Caspase inhibitors do not repress ATP-induced SAPK activity. The caspase-3-like inhibitor DEVD-cho (300 µM) or the caspase-1-like inhibitor YVAD-cmk (200 µM) was added to BAC1 macrophages 30 min prior to challenge with ATP. 5 mM ATP was added, and cells were incubated for 30 min at 37 °C, washed with ATP-free medium containing inhibitors, and incubated for an additional 60 min. Cells were processed, and SAPK activity was determined as described in the legend to Fig. 1. con, control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Despite strong evidence that the P2X7 receptor regulates macrophage apoptosis, IL-1 release, and killing of intracellular mycobacteria, little is known regarding the signaling pathways that mediate these processes. This study implicates SAPK as a novel P2X7 receptor effector and represents the first demonstration of stress-activated kinase signaling by this particular ATP-gated ion channel. More important, this activation of SAPK was triggered using brief pulses of extracellular ATP added to tissue culture medium containing physiologic levels of divalent cations, conditions designed to approximate the environment of a tissue macrophage.

Although millimolar levels of extracellular ATP were required to elicit SAPK activation, the pharmacological and nucleotide selectivity data suggest that the P2X7 receptor alone specifically mediates this effect. Although oxidized ATP has been extensively used as an effective P2X7 receptor antagonist, recent data suggest that this agent has multiple P2 receptor targets (40, 41). For this reason, we also tested KN-62, an isoquinoline derivative and Ca²⁺/calmodulin-dependent protein kinase II inhibitor first shown by Blanchard et al. (42) to inhibit P2X7-dependent signaling. Gargett and Wiley (43) demonstrated that this inhibition of P2X7 receptor function does not involve Ca²⁺/calmodulin-dependent protein kinase II. We have documented a strong species dependence on KN-62 sensitivity, with the human P2X7 receptor exhibiting an IC₅₀ of 30 nM, the mouse receptor being ~10-fold lower less sensitive, and the rat P2X7 receptor being virtually unaffected by this compound (33). Thus, inhibition by both oxidized ATP and KN-62 strongly suggests that the ATP-induced SAPK activity is mediated by the P2X7 receptor. The nucleotide selectivity series also supports a role for this receptor as the sole mediator of SAPK activation. The requirement for millimolar ATP and the ability of submillimolar 3'-O-(4-benzoyl)benzoyl-ATP to mimic this effect of ATP typify the agonist profile of the P2X7 receptor. Finally, the inability of a caspase-1 (interleukin-converting enzyme) inhibitor or a neutralizing anti-TNF- antibody to attenuate stimulation of SAPK by extracellular ATP argues against a role for secreted IL-1 or TNF- as an indirect mediator of this ATP effect.

Although wild-type HEK293 cells natively express both P2Y1 and P2Y2 receptors (45), ATP pulsing did not trigger SAPK activation in these cells. Rather, heterologous expression of the P2X7 receptor in HEK293 cells appears necessary and sufficient to mediate the activation of SAPK by extracellular ATP in these fibroblast-like cells. This suggests that the biochemical pathway linking the channel/pore to SAPK is not restricted to macrophages. Whether P2X7 receptor expression is sufficient to reconstitute this pathway in other cellular backgrounds remains to be determined. Given recent data suggesting that the P2X7 receptor channel and the induced nonselective pore may be biochemically distinct entities (10), the SAPK-inducing signal could emanate from either the channel or the pore. It would be interesting to test whether SAPK can be activated by maitotoxin, a pore-inducing toxin that appears to couple to the same cytolytic pore as the P2X7 receptor (44).

P2X7 receptor ligation activates caspase-1 and caspase-3 in murine microglial cells and dendritic cells (18, 19), and our study confirms these findings in BAC1 macrophages. Caspase inhibitors abrogate induction of NF-B activity by P2X7 agonists, so caspases have been proposed to mediate P2X7 receptor-dependent NF-B induction (20). We hypothesized that the MEKK1/MEK/SAPK pathway might play a role in this signaling cascade because MEKK1 regulates activation of both SAPK and NF-B (24, 48). Moreover, caspase-3-dependent cleavage of MEKK1 into a 91-kDa fragment increases its kinase activity and could explain why P2X7-dependent NF-B induction is sensitive to caspase inhibition (27, 36). However, two lines of investigation strongly indicate that activation of caspase-1- and caspase-3-like proteases was not an obligatory step in the signaling pathways coupling P2X7 receptors to SAPK stimulation. First, SAPK activation preceded the accumulation of active caspase-3 by several hours. Second, inhibitors of either caspase-3- or caspase-1-like proteases had no effect on SAPK activation. These peptide inhibitors blocked caspase activity as reflected by their effects on -fodrin breakdown and IL-1 release, indicating that sufficient concentrations of inhibitor were used to inactivate both proteases. One interpretation of these results is that SAPK lies upstream of caspase activation; and indeed, SAPK has been shown to induce caspase activation (46). Alternatively, independent signaling pathways may link P2X7 receptors to the activation of SAPK and the various caspases. Induction of apoptosis itself is a form of cell stress that may trigger SAPK signaling without altering the execution of the cell death program (28, 47).

The mechanism by which P2X7 receptors trigger activation of the SAPK signaling cascade is unclear. Ca²⁺-dependent activation of SAPK has been described (50). However, a simple rise in intracellular Ca²⁺ is an unlikely explanation for the ATP-mediated SAPK activation because UTP did not trigger SAPK despite its ability to induce a sustained increase in Ca²⁺ in BAC1 macrophages via activation of G protein-coupled P2Y2 receptors (34). Oxidative stressors are potent inducers of SAPK activity (53), and reactive oxygen intermediates accumulate in microglial cell stimulated by P2X7 receptor agonists (20). ATP-induced activation of SAPK in BAC1 macrophages was unaffected by the reducing agent N-acetylcysteine (data not shown), making a role for reactive oxygen intermediates less likely. Depletion of bulk intracellular K⁺ is implicated in SAPK and caspase activation as well as apoptosis and necrosis (29, 30, 51, 54). Consistent with a possible role for K⁺ efflux in mediating P2X7 receptor activation of SAPK, inhibiting K⁺ efflux in BAC1 macrophages (by replacing extracellular Na⁺ with K⁺) attenuated, but did not eliminate the ATP-mediated SAPK activity (data not shown). Unequivocal interpretation of these experiments is difficult because this manipulation of the extracellular ionic conditions exerts striking effects on both the efficacy and potency of ATP as a P2X7 receptor agonist (52).

The identification of SAPK as a P2X7 receptor effector opens new avenues for identifying the biochemical events that underlie processes involving the P2X7 receptor such as M. tuberculosis killing, apoptosis, and IL-1 release. Future studies must address both the mechanism by which the P2X7 receptor couples to SAPK and the physiologic consequences of activating this signaling pathway.

	ACKNOWLEDGEMENTS

We are grateful to Reza Beigi and Lalitha Gudipaty for helpful discussions.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant GM36387.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.

Supported by Medical Scientist Training Program Grant GM07250-24 from the National Institutes of Health.

^§ To whom correspondence should be addressed: Dept. Physiology and Biophysics, School of Medicine E565, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4970. Tel.: 216-368-5523; Fax: 216-368-3952; E-mail: gxd3@po.cwru.edu.

Published, JBC Papers in Press, June 14, 2000, DOI 10.1074/jbc.M002770200

	ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide; IL-1, interleukin-1; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; TNF-, tumor necrosis factor-; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MEKK, MEK kinase; GST, glutathione S-transferase; ELISA, enzyme-linked immunosorbent assay; DEVD-cho, Asp-Glu-Val-Asp-cho; YVAD-cmk, Tyr-Val-Ala-Asp-chloromethyl ketone; HEK, human embryonic kidney; DTT, dithiothreitol; cho, aldehyde; NFAT, nuclear factor of activated T cells; TRAF, tumor necrosis factor receptor-associated factor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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