1Department of Biomolecular Chemistry and 2Program in Molecular and Cellular Pharmacology, University of Wisconsin Medical School, and 3Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53706
Submitted 30 September 2003 ; accepted in final form 8 December 2003
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ABSTRACT |
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nucleotide receptors; mitogen-activated protein kinases; nuclear factor-B; monocytes/macrophages; cytokines
These effects are initiated via extracellular nucleotide interaction with a variety of cell surface nucleotide receptors in multiple cell types (13, 36, 38). In this regard, the P2 class of the nucleotide receptors, which preferentially binds di- and triphosphonucleotides, has been classified into two separate families: the P2Y receptors, which are seven-transmembrane-spanning heterotrimeric G protein-coupled receptors, and the P2X receptors, which serve as ATP-gated plasma membrane cation channels (1, 6, 36). One member of the P2X receptor family, P2X7, is highly expressed in cells of immune and hematopoietic lineage (6, 18, 33). When P2X7 is activated by its natural ligand ATP or the pharmacological agonist BzATP, the cell membrane undergoes rapid depolarization (28, 52), resulting in an influx of calcium and sodium and the efflux of potassium (28, 29). Prolonged activation of P2X7 promotes the formation of a nonselective pore that allows for the bidirectional passage of metabolites of <900 Da, leading to cytoplasmic leakage and the generation of ion fluxes that are believed to cause cell apoptosis (19, 30). The mammalian P2X7 is 595 amino acids in length and possesses the longest COOH terminus among all P2X receptors (52, 53). This COOH-terminal feature appears to endow the receptor with the capacity to promote pore formation and apoptosis (2, 14) as well as the ability to initiate various signaling cascades (2, 6, 24, 32, 34), possibly by serving as a docking site for various lipid and protein effectors (15, 45, 56).
Although the signaling events associated with the copresentation of nucleotides and LPS in the generation of these immune responses remain to be clearly elucidated (26, 32, 54), there is evidence supporting a role for the mitogen-activated protein (MAP) kinases. These enzymes are a highly conserved family of protein serine/threonine kinases and include the extracellular signal-regulated kinases ERK1/2, the c-Jun NH2-terminal kinases JNK1/2, and the p38 stress-activated protein kinases (21, 47, 49, 50). These kinases can trigger the nuclear accumulation and activity of various transcription factors, such as NF-B, NFAT, ATF2, Ets, and c-Jun, which can modulate cytokine and inflammatory mediator expression (4, 7, 9, 42). Recent data suggest that the p38 MAP kinase is critical for LPS-induced inducible NO synthase (iNOS) expression, NO production, and the activation of NF-
B DNA-binding activity in macrophages (11, 12). Interestingly, P2X7 signaling is associated with an increase in LPS-stimulated IL-1
processing, TNF-
release, and iNOS expression as well as NO and IL-6 production in monocytes and macrophages (3, 4, 7, 8, 51, 54).
Multiple studies have shown that the expression of several cytokine genes including TNF-, IL-6, and IL-1
is associated with NF-
B activation (3, 4, 9, 11, 12, 31). Because LPS and P2X7 receptor agonists can modulate NF-
B DNA-binding activity, this parameter is a potential point of convergence in the cross talk between the signaling events initiated by LPS and extracellular nucleotides. NF-
B is a homo- or heterodimeric transcription factor whose activity is regulated by the binding of an inhibitory protein, I
B (16). Upon serine phosphorylation, I
B is targeted for proteasome-mediated degradation, allowing the NF-
B protein complex to translocate into the nucleus, where it can bind to regulatory elements present in various gene promoter regions (31). Of relevance to the present studies, both LPS (55) and BzATP (2) have been shown to stimulate the degradation of one of the isoforms of I
B, namely, I
B
. Accordingly, both LPS and BzATP can promote the activation of NF-
B DNA-binding activity in numerous myelocytic cell types, including phagocytes (57), although the mechanism(s) by which these agents may interact to coordinately activate NF-
B is undefined.
Although extracellular nucleotides affect LPS-mediated inflammatory mediator production in macrophages, monocytes, and microglial cells via P2X7 (22, 23), the intracellular signaling pathways proximal to P2X7 that lead to regulation of LPS-induced inflammatory mediator production in these cells have yet to be fully established. However, two key events thought to be important in governing LPS-mediated cytokine generation are the activation of members of the MAP kinase and NF-B families. Hence, the present studies were undertaken to test the idea that there exists cross talk between LPS- and nucleotide receptor-dependent activation of the MAP kinases and NF-
B.
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MATERIALS AND METHODS |
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Cell culture. Murine RAW 264.7 macrophage cells obtained from ATCC (Rockville, MD) were grown to 80% confluency and routinely passaged in RPMI 1640 medium containing 5% cosmic calf serum and 100 U/ml penicillin/streptomycin (Life Technologies, Gaithersburg, MD). Human monocytic THP-1 cells obtained from ATCC were grown to
80% confluency in RPMI 1640 medium containing 10% fetal bovine serum and 100 U/ml penicillin/streptomycin. RAW 264.7 and THP-1 cells were cultured routinely in 100-mm Falcon plates (Becton Dickinson, Franklin Lakes, NJ). Before each experiment, the cells were plated overnight in Falcon 6 (3 x 105 cells/well)-, 12 (5 x 105 cells/well)-, or 24-well plates (1.5 x 105 cells/well). The next day, the cells were treated as indicated. These treatments were terminated by removing the medium and washing the cells twice with cold Hanks' balanced salt solution containing 1 mM Na3VO4.
Immunoblotting for MAP kinases and IB
. Whole cell lysates were prepared by lysing RAW 264.7 and THP-1 cells, plated as indicated in Cell culture, in SDS-PAGE sample buffer (20 mM Tris, 2 mM EDTA, 1 mM Na3VO4, 2 mM DTT, 2% SDS, and 20% glycerol). Protein content was determined by using the Micro-BCA protein assay (Pierce Biochemical, Rockford, IL). Equal amounts of protein were loaded per lane and resolved by 10% SDS-PAGE as described earlier (37). Proteins were transferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA) and blocked in 5% dry nonfat milk-TBST (10 mM Tris·HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20) either overnight at 4°C or for 1 h at 37°C. Anti-active ERK antibodies that recognize the dually tyrosine and threonine phosphorylated and thus enzymatically active forms of ERK1/2 were used at a dilution of 1:5,000. Anti-I
B
antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were diluted 1:1,000 in 5% milk-TBST. Anti-active p38 antibodies were used at a final dilution of 1:4,000 in 5% milk-TBST. Anti-active JNK antibodies were employed at a final concentration of 1:5,000 in 0.1% IgG and protease-free BSA-TBST. The membranes were incubated with the primary antibody for 1 h at 37°C or, when anti-active JNK antibodies were used, for 2 h at room temperature. The immunoreactive bands were visualized by using secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology) and Supersignal West Pico as the chemiluminescent substrate (Pierce). To confirm equal protein loading, membranes were stripped at 70°C for 30 min with a buffer consisting of 62.5 mM Tris·HCl (pH 6.7), 2% SDS, and 100 mM DTT. The immunoblots were reblocked in 5% milk-TBST, followed by incubation with antibodies that react with both active and inactive forms of ERK1/2 (Upstate Biotechnology, Lake Placid, NY). Alternatively, anti-Grb2 antibodies (Santa Cruz Biotechnology; 1:1,000 in 5% milk-TBST) were used concomitantly to evaluate Grb2 levels as an indicator of the consistency of protein loading.
Measurement of iNOS and NO. Murine RAW 264.7 cells were treated with LPS or BzATP at the concentrations indicated. The NO levels in the medium were determined by evaluating the concentration of its stable breakdown product, nitrite, using the Greiss reagent as described previously (41). For iNOS determination, the medium was removed, the cells were washed, and whole cell lysates were prepared as described in Immunoblotting for MAP kinases and IB
. The levels of iNOS were measured by resolving the proteins by 10% SDS-PAGE and immunoblotting with anti-iNOS antibodies (1:2,000) (Transduction Laboratories, Lexington, KY).
Electrophoretic mobility shift assays. Nuclear extracts were prepared from RAW 264.7 macrophages grown in 100-mm Falcon plates, and gel mobility shift assays were performed as described previously (16). Briefly, cells were treated with the control buffer (20 mM HEPES) or with LPS (10 ng/ml) or BzATP (100 µM) for 60 min, either alone or in combination. The double-stranded oligonucleotide probe encoding two consensus NF-B DNA-binding sites (5'-GATCCAAGGGACTTTCCATGGATCCAAGGGGACTTTCCATG-3') was labeled by using [
-32P]ATP and T4 polynucleotide kinase. The cells were lysed, and 5 µg of nuclear protein extract were incubated with 1 x 105 cpm of the labeled oligonucleotide probe at room temperature for 20 min. The proteins were then separated on a 6% nondenaturing polyacrylamide gel and analyzed by autoradiography.
Measurement of Ras activation. Murine RAW 264.7 macrophages were treated with LPS and/or BzATP as described in RESULTS, and the cells were then mixed with lysis buffer containing 10% glycerol, 50 mM Tris·HCl (pH 7.4), 200 mM NaCl, 1% NP-40, and 2 mM MgCl2. The cell lysates were then precleared with glutathione-agarose beads (Pierce), using a 10-min incubation at 4°C, followed by centrifugation to remove the beads. The precleared supernatants were incubated with glutathione-agarose beads conjugated to a glutathione-S-transferase (GST) fusion protein of the Ras-binding domain of Raf (Upstate Biotechnology) for 30 min at 4°C. The beads were washed three times with lysis buffer, and active Ras proteins were eluted with SDS-PAGE sample buffer and boiled for 5 min. The proteins were separated by SDS-PAGE and immunoblotted by using anti-Ras antibodies (Transduction Laboratories).
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RESULTS |
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Activation of NF-B in response to LPS and nucleotides. To further delineate the mechanism by which P2X7 agonists increase iNOS expression and NO production when coadministered with LPS, we evaluated the effects of these agents on the activation of NF-
B (Fig. 2), which is a transcription factor important for controlling the expression of many cytokine and inflammatory mediator genes, including iNOS. Treatment of RAW 264.7 macrophages for 60 min with either LPS (10 ng/ml) or the P2X7 agonist BzATP alone resulted in an increase in NF-
B DNA-binding activity, whereas cotreatment of the cells with both LPS and BzATP promoted a substantially elevated level of NF-
B-associated DNA-binding activity (Fig. 2). These results are consistent with the observations that P2X7 activation augments LPS-induced iNOS expression and NO production and suggest that enhanced NF-
B activity may contribute to the mechanism of this effect.
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Expression of IB
levels in response to treatment with LPS, BzATP, and the MEK antagonist U0126. Because I
B
is a regulatory protein that binds to and inhibits NF-
B, and given that concurrent stimulation of RAW 264.7 macrophages with LPS and BzATP leads to increased NF-
B DNA-binding activity, we evaluated the kinetics of I
B
degradation and reappearance in murine macrophages (Fig. 3A). Upon simultaneous treatment of RAW 264.7 macrophages with LPS and BzATP for 15 min, I
B
levels were found to be nearly undetectable, and this early effect appears similar to that observed with LPS treatment alone. However, coadministration of LPS and BzATP for longer times revealed differences in the kinetics of I
B
reappearance compared with LPS treatment alone (Fig. 3A); i.e., the reestablishment of I
B
levels was not evident until 60 min after incubation of the cells with LPS plus BzATP, whereas I
B
levels returned to baseline more rapidly (within 3045 min) after treatment with LPS alone.
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Because MAP kinase pathways have been reported to be involved in controlling the production of inflammatory mediators such as NO, and given that concurrent treatments with LPS and BzATP influence iNOS expression (Fig. 1), NF-B DNA-binding activity (Fig. 2), and I
B
reappearance (Fig. 3A), we evaluated the effect of the MEK1/2 inhibitor U0126 on I
B
reappearance in RAW 264.7 cells to determine the possible role of the MEK/ERK pathway in arbitrating these responses. To this end, murine RAW 264.7 macrophages were treated with LPS in the presence and absence of U0126 (Fig. 3B). U0126 by itself had no effect on I
B
degradation (data not shown); however, incubation of the cells with the MEK inhibitor delayed the reappearance of I
B
after LPS treatment, which is similar to the effects observed after treatment with LPS plus BzATP. Again, although the levels of I
B
at the 15 min time point were similar in LPS-treated cells, with or without the addition of U0126 (Fig. 3B), a delay in the reappearance of I
B
was clearly observed from 30 to 120 min when the LPS-treated cells were coincubated with U0126. These differences do not appear to arise from variations in protein loading because the levels of the adaptor protein Grb2 were comparable in all lanes. Therefore, these observations support the intriguing notion that the dynamics of I
B
expression/turnover may be closely related to MEK1/2-dependent signaling pathways.
Influence of LPS and the P2X7 agonist BzATP on MAP kinase activation. The ability of P2X7 to influence the activation of various MAP kinase pathways may represent a key mechanism whereby LPS- and nucleotide-stimulated signal transduction pathways interact in macrophages to control iNOS expression and NO production. Thus we tested the ability of P2X7 receptor agonists to modulate LPS-stimulated MAP kinase activation in RAW 264.7 macrophages (Fig. 4). Stimulation of murine macrophages with the P2X7 ligand BzATP or with LPS was observed to lead to a time-dependent increase in the activation of ERK1/2 (Fig. 4, top), JNK1/2 (middle), and p38 (bottom) MAP kinases.
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MAP kinase activation in response to cotreatment with LPS and the P2X7 agonist BzATP. Because LPS and the P2X7 receptor agonist BzATP stimulated the activation of multiple MAP kinases (Fig. 4), we examined the effect of concurrent administration of LPS and P2X7 receptor agonists on MAP kinase activation (Fig. 5). This analysis was especially critical given the apparent conflicting observations that although BzATP can activate the ERK1 and ERK2 MAP kinases (Fig. 4), this P2X7 agonist appears to have overlapping activity with the MEK antagonist U0126 in that it can also promote a delay in the reappearance of IB
levels after LPS stimulation (Fig. 3). Thus, in these studies, RAW 264.7 cells were incubated for various times with LPS and/or BzATP. As shown in Fig. 5A, we observed a marked inhibition in the levels of ERK1/2 activation upon cotreatment of macrophages with BzATP and LPS compared with cells treated with either agonist individually. This effect is more evident at the earlier time points (2, 5, and 7.5 min) and progressively diminishes at the later time point (30 min). Interestingly, the ability of BzATP to alter the kinetics of ERK activation when coadministered with LPS may be linked to the capacity of BzATP and the MEK antagonist U0126 to exert similar effects on the kinetics of LPS-induced I
B
expression/degradation (Fig. 3).
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In contrast to the effects of nucleotides on ERK activation, an increase in the activation of the JNK1/2 and p38 MAP kinases (Fig. 5B) was observed in response to cotreatment of murine RAW 264.7 macrophages with BzATP and LPS compared with cell treatment with BzATP or LPS alone. These differential effects on various members of the MAP kinase family were not restricted to murine macrophages but were also detected with human THP-1 monocytes (Fig. 5C). Treatment with LPS and BzATP alone for 10 min can activate ERK1/2, JNK1/2, and p38 MAP kinases in the THP-1 cells. However, simultaneous exposure of these cells to LPS and BzATP for 10 min resulted in an attenuation in ERK1/2 activation, whereas an additive increase in the activation of JNK1/2 and p38 MAP kinases was observed. These data indicate that concomitant exposure of macrophages to LPS and nucleotides results in the coordinate regulation of the MAP kinases and NF-B/I
B.
Kinetics of Ras activation in response to treatment with LPS and nucleotides. To begin to assess the mechanism by which nucleotide and LPS cotreatment of macrophages suppresses ERK1/2 activation, we evaluated the influence of LPS and BzATP on the small molecular weight G protein Ras in murine RAW 264.7 macrophages, given that Ras is a known upstream activator of these ERKs. The cells were treated with LPS or BzATP alone or together for various times. As shown in Fig. 6A, BzATP potently stimulates Ras activation as early as 2 min after treatment, which is an effect that persists for at least 15 min. In contrast, stimulation of macrophages with LPS alone results in a slower and weaker activation of Ras, consistent with reports suggesting that LPS may also promote ERK activation via a Ras-independent mechanism (5, 44, 48). Interestingly, as shown in Fig. 6, A and B, upon costimulation with LPS and BzATP, we observed a marked reduction in BzATP-mediated Ras activation. These data suggest that events proximal to Ras activation are involved in the cross talk between LPS- and nucleotide receptor-mediated signaling.
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DISCUSSION |
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To evaluate the mechanism(s) by which NF-B activation is modulated by LPS and P2X7 agonists, we examined MAP kinase activation and its relationship to regulation of the NF-
B inhibitory protein I
B
. In terms of MAP kinase activation, we found that macrophage stimulation with BzATP alone led to a rapid activation (within 2 min) of both Ras and ERK1/2, whereas treatment with LPS alone led to a slower activation (within 515 min) of ERK1/2 and minimal activation of Ras. Interestingly, BzATP-stimulated Ras activation was attenuated in the presence of LPS, and this corresponded to a decrease in the activation of ERK1/2 at early time points (210 min posttreatment) compared with that initiated by LPS or BzATP alone. This attenuation of ERK activation may result from competition between these two systems for the activation or membrane localization of upstream regulators of Ras or Raf, and/or it may result from cross talk between various kinases, phosphatases, and other effector molecules that are uniquely regulated by LPS- and nucleotide receptor-dependent processes. For example, the P2X7 COOH terminus is known to contain an LPS-binding domain and multiple protein- and lipid-interaction motifs that may affect receptor-protein/lipid interactions (15, 56). Accordingly, LPS regulation of P2X7 coupling to Ras-dependent pathways may differentially influence MAP kinase activation and I
B/NF-
B-associated signaling.
The data presented here also indicate that macrophages cotreated with LPS and BzATP display a sustained loss of IB
, similar to the effects observed when the cells were treated with LPS and the MEK inhibitor U0126. These data suggest that NF-
B may exist longer in a transcriptionally active state when ERK activation is attenuated in the presence of P2X7 ligands or the MEK inhibitor. The possible negative role of ERK-1 and ERK-2 activation on NF-
B action is in accordance with earlier results revealing that inhibition of MEK/ERK pathway leads to enhanced mediator production in macrophage-like cells (55).
Because IB
degradation is central to NF-
B activation, and because the control of I
B
levels can occur at the level of either transcription or degradation, an alteration in the degradation or synthesis of I
B
would influence the length of time over which NF-
B is active or functional. In this regard, our data indicate that costimulation of macrophages with LPS and BzATP results in a delay in I
B
reappearance compared with LPS stimulation alone, and that costimulation of macrophages with LPS and U0126 also impedes the reappearance of I
B
. One explanation for these results is that LPS can induce a more rapid degradation of I
B
compared with BzATP and that exposing the cells to both LPS and BzATP leads to the additivity or convergence of these pathways, resulting in a sustained suppression of I
B
levels and thus a prolonged activation of NF-
B. Furthermore, our data suggest that a reduction in MEK/ERK activation may either accelerate I
B
degradation or reduce the efficiency of I
B
gene expression. Although previous reports indicate that MAP kinase activation may be involved in the activation of NF-
B (4, 10), very little is known about the interaction of MEK/ERK pathway with I
B
/NF-
B.
Another explanation for the observed IB
effects stems from the possible influence of LPS and BzATP on the function of NIK (NF-
B-inducing enzyme) and MEKK signaling molecules. Dhawan and Richmond (17) reported that transfection of human melanoma cells with either kinase-inactive mutants of NIK or dominant negative MEKK1 and ERK1/2 constructs results in decreased basal promoter activity of NF-
B, suggesting the potential for cross talk between the MAP kinases and the NF-
B pathways. Because the I
B
gene is regulated by NF-
B, a reduction in MEK/ERK activity by the copresentation of LPS and BzATP may attenuate I
B
expression. In addition, Nakano et al. (43) reported that MEKK1 overexpression in HEK-293 cells regulates I
B kinase
(IKK
), and that NIK overexpression stimulates the activity of both IKK
and IKK
, suggesting that MEKK1 activation can influence IKK-associated signaling pathways. Furthermore, it is interesting to note that both IKK
and IKK
proteins contain a regulatory loop that is similar to one found in the MEK family of proteins (35). These structural similarities indicate that the MEK pathway may alter the activity of IKK and influence the phosphorylation and turnover of I
B
. Altogether, these previous studies along with the present data support the idea of the potential cross talk between the MEK/ERK and the I
B/NF-
B signal transduction pathways following cotreatment with LPS and P2X7 receptor agonists such as BzATP.
Besides the contribution of MEK/ERK in this system, the potentiation of p38 and JNK1/2 by the combined actions of LPS and P2X7 receptor agonists may also control the production of inflammatory mediators. For example, p38 has been reported to be important for the generation of NO in macrophages (11, 12, 55), and Meng et al. (40) demonstrated that MAP kinase-activated protein kinase 2 (MAPKAP2), which is a downstream target of p38, can lead to increased mediator production. Conversely, addition of the MEK inhibitor U0126 to RAW 264.7 cells increases the production of NO (55), suggesting that the MEK/ERK pathway may serve as a negative feedback mechanism for the control of inflammatory mediator production. Because the cotreatment of macrophages and monocytes with LPS and BzATP results in the increased activation of p38 and JNKs, and because p38 activation is critical for the generation of NO (11, 12, 34, 55), it is possible that the increase in the p38 activation is a dominant pathway and can block ERK1/2 activation. These findings are consistent with the concept that inhibition of ERK1/2, by either cotreatment with LPS and BzATP or the addition of a MEK inhibitor, alters the regulation of IB/NF-
B signal transduction pathways.
In summary, this present work provides evidence supporting a mechanism for the regulation of inflammatory mediator production by macrophages after exposure to LPS and extracellular nucleotides, such as that encountered in the inflammatory microenvironment. These agents can lead to the coordinate control of the MAP kinases and NF-B/I
B
-dependent pathways, thereby altering inflammatory mediator synthesis and release. Delineating the interaction of the MAP kinases with the I
B/NF-
B network in LPS and nucleotide action may offer valuable insight with regard to discovering new therapeutic targets and provide a better understanding of the signaling mechanisms associated with endotoxemia and septic shock.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by National Institutes of Health Grants GM-53271, HL-56396, and AI-50500. J. J. Watters was supported by National Research Service Award F32-CA-81733. Z. A. Pfeiffer and J. A. Sommer were supported by National Institutes of Health Biotechnology Training Program T32-GM-08349.
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FOOTNOTES |
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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.
* M. Aga and J. J. Watters contributed equally to this work.
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