The nucleotide receptor P2X7 has been shown to modulate LPS-induced macrophage production of numerous inflammatory mediators. Although the C-terminal portion of P2X7 is thought to be essential for multiple receptor functions, little is known regarding the structural motifs that lie within this region. We show here that the P2X7 C-terminal domain contains several apparent protein-protein and protein-lipid interaction motifs with potential importance to macrophage signaling and LPS action. Surprisingly, P2X7 also contains a conserved LPS-binding domain. In this report, we demonstrate that peptides derived from this P2X7 sequence bind LPS in vitro. Moreover, these peptides neutralize the ability of LPS to activate the extracellular signal-regulated kinases (ERK1, ERK2) and to promote the degradation of the inhibitor of κB-α isoform (IκB-α) in RAW 264.7 macrophages. Collectively, these data suggest that the C-terminal domain of P2X7 may directly coordinate several signal transduction events related to macrophage function and LPS action.
Extracellular nucleotides regulate macrophage function via P2X nucleotide receptors that form ligand-gated ion channels. In particular, P2X7 activation is characterized by pore formation, membrane blebbing, and cytokine release. P2X7 is also linked to mitogen-activated protein kinases (MAPK) and Rho-dependent pathways, which are known to affect cytoskeletal structure in other systems. As cytoskeletal function is critical for macrophage behavior, we have tested the importance of these pathways in actin filament reorganization during P2X7 stimulation in RAW 264.7 macrophages. We observed that the P2X7 agonists adenosine 5'-triphosphate (ATP) and 3'-O-(4-benzoylbenzoyl) ATP (BzATP) stimulated actin reorganization and concomitant membrane blebbing within 5 min. Disruption of actin filaments with cytochalasin D attenuated membrane blebbing but not P2X7-dependent pore formation or extracellular-regulated kinase (ERK)1/ERK2 and p38 activation, suggesting that these latter processes do not require intact actin filaments. However, we provide evidence that p38 MAPK and Rho activation but not ERK1/ERK2 activation is important for P2X7-mediated actin reorganization and membrane blebbing. First, activation of p38 and Rho was detected within 5 min of BzATP treatment, which is coincident with membrane blebbing. Second, the p38 inhibitors SB202190 and SB203580 reduced nucleotide-induced blebbing and actin reorganization, whereas the MAPK kinase-1/2 inhibitor U0126, which blocks ERK1/ERK2 activation, had no discernable effect. Third, the Rho-selective inhibitor C3 exoenzyme and the Rho effector kinase, Rho-associated coiled-coil kinase, inhibitor Y-27632, markedly attenuated BzATP-stimulated actin reorganization and membrane blebbing. These data support a model wherein p38- and Rho-dependent pathways are critical for P2X7-dependent actin reorganization and membrane blebbing, thereby facilitating P2X7 involvement in macrophage inflammatory responses.
Macrophage activation is critical in the innate immune response and can be regulated by the nucleotide receptor P2X 7 . In this regard, P2X 7 signaling is not well understood but has been implicated in controlling reactive oxygen species (ROS) generation by various leukocytes. Although ROS can contribute to microbial killing, the role of ROS in nucleotide-mediated cell signaling is unclear. In this study, we report that the P2X 7 agonists ATP and 3′-O-(4-benzoyl) benzoic ATP (BzATP) stimulate ROS production by RAW 264.7 murine macrophages. These effects are potentiated in lipopolysaccharide-primed cells, demonstrating an important interaction between extracellular nucleotides and microbial products in ROS generation. In terms of nucleotide receptor specificity, RAW 264.7 macrophages that are deficient in P2X 7 are greatly reduced in their capacity to generate ROS in response to BzATP treatment (both with and without LPS priming), thus supporting a role for P2X 7 in this process. Because MAP kinase activation is key for nucleotide regulation of macrophage function, we also tested the hypothesis that P2X 7 -mediated MAP kinase activation is dependent on ROS production. We observed that BzATP stimulates MAP kinase (ERK1/ ERK2, p38, and JNK1/JNK2) phosphorylation, and that the antioxidants N-acetyl-cysteine and ascorbic acid strongly attenuate BzATP-mediated JNK1/JNK2 and p38 phosphorylation but only slightly reduce BzATP-induced ERK1/ERK2 phosphorylation. These studies reveal that P2X 7 can contribute to macrophage ROS production, that this effect is potentiated upon lipopolysaccharide exposure, and that ROS are important participants in the extracellular nucleotide-mediated activation of several MAP kinase systems.
Sigma receptors once considered as a class of opioid receptors are now regarded as unique orphan receptors, distinguished by the ability to bind various pharmacological agents such as the progesterone (steroid), haloperidol (anti-psychotic), and drugs of abuse such as cocaine and methamphetamine. The sigma-1 receptor is a 223 amino acid protein, proposed to have two transmembrane segments. We have developed a scheme for the purification of the guinea pig sigma-1 receptor following overexpression in Escherichia coli as a maltose binding protein (MBP) fusion and extraction with Triton X-100. Affinity chromatography using an amylose column and Ni2+ affinity column was used to purify the sigma-1 receptor. The sigma-1 receptor purified by this method is a 26 kDa polypeptide as assessed by SDS-PAGE, binds sigma ligands with high affinity and can be specifically photoaffinity labeled with the sigma-1 receptor photoprobe, [125I]-iodoazidococaine. Ligand binding using [3H]-(+)-pentazocine indicated that approximately half of the purified protein in Triton X-100 bound to radioligand. The MBP-sigma-1 receptor and the sigma-1 receptor in 0.5% triton were maximally stable for approximately two weeks at -20 degrees C in buffer containing 30% glycerol.
The nucleotide receptor P2X7 is expressed by most leukocytes and initiates signaling events that amplify numerous LPS responses. We tested the hypothesis that loss-of-function polymorphisms in the human P2X7 gene predispose to the production of an anti-inflammatory mediator balance. Accordingly, we developed a novel P2X7 pore assay in whole blood that magnifies the activity from wild-type alleles and preserves the gene dosage effect for the 1513 C polymorphism (AA, 69 ± 4; AC, 42 ± 4; and CC, 6 ± 1-fold stimulation). Thirty of 200 healthy individuals were identified as having low P2X7 pore activity. Seven low pore subjects were 1513 CC, 3 and 11 participants had the other known variants 946 GA and 1729 TA respectively; the remaining 9 volunteers likely have novel polymorphisms. Because platelets are a large source of extracellular ATP during inflammation, whole blood was treated ex vivo with Salmonella typhimurium LPS in the absence of exogenous nucleotides. LPS-stimulated whole blood from individuals in the low pore activity group generated reduced plasma levels of TNF-α (p = 0.036) and higher amounts of IL-10 (p < 0.001) relative to the high pore controls. This reduction in the TNF-α to IL-10 ratio persisted to at least 24 h and is further decreased by cotreatment with 2-methylthio-ATP. The ability of P2X7 polymorphisms to regulate the LPS-induced TNF-α to IL-10 ratio suggests that 15% of healthy adults may exhibit anti-inflammatory mediator responses during major infectious perturbations of the immune system, which can be predicted by P2X7 pore activity.
Background: Innate immune response amplification is achieved by leukocyte expression of the purinergic nucleotide receptor P2X 7 , an extracellular nucleotidegated pore. Previously, low P2X 7 pore activity in whole blood was associated with loss-of-function genotypes in correlation with a decreased ratio of lipopolysaccharidestimulated tumor necrosis factor-␣ to interleukin-10, of relevance to a variety of infectious and inflammatory disorders. We hypothesized that evaluation of participants with discordance between the P2X7 genotype and pore status would disclose additional alleles, linkage disequilibrium, and novel functional correlates of genotype to phenotype. Methods: Comparison of whole-blood pore results with restriction fragment length polymorphism data for known loss-of-function genotypes from 200 healthy participants optimized the diagnostic threshold for low pore activity by ROC curve analysis. We identified novel alleles and inferred haplotypes by sequencing outlier genomic templates and by linkage analysis. Results: With a refined threshold of low activity, a normal pore result had only a 2% probability of association with known loss-of-function variants. By contrast, the positive predictive value of low pore activity was 59% for identifying known alleles. DNA samples from this discordant group contained 28 P2X7 sequence variations. Linkage analysis demonstrated that A1513C, T1729A, and G946A are inherited independently from one another, although these loss-of-function variants are
Macrophages express several lipopolysaccharide (LPS) binding proteins and are potently activated by LPS to produce inflammatory mediators. Recent studies have shown that receptors for exogenous nucleotides (P2X and P2Y purinergic receptors) can modulate macrophage production of TNF-alpha, IL-1beta and nitric oxide (NO) following LPS exposure. Macrophages and LPS-stimulated monocytes express elevated levels of P2Y1, P2Y2 and P2X7 mRNA, suggesting that both P2Y and P2X receptors can contribute to LPS-induced pathophysiology. In addition, oxidized-ATP treatment (which inhibits P2X7) of macrophages blocks LPS-induced NO production, NF-kappaB and ERK-1/2 activation. Also, an LPS-binding domain located in the P2X7 C-terminus appears important for receptor trafficking/function. Moreover, the purinergic receptor ligand 2-MeS-ATP attenuates LPS-induced cytokine and NO production in vivo and ex vivo. These data suggest that P2X7 and certain P2Ys are linked to LPS effects, although their relative contribution in vivo is unclear. Accordingly, we tested the capacity of several adenine nucleotides to modulate LPS-induced mortality in mice. We found that the P2X7-directed ligand BzATP was unable to prevent LPS-induced death, whereas 2-MeS-ATP and 2-Cl-ATP, which bind to multiple P2X and P2Y receptors were able to protect mice from LPS-induced death. These data suggest that the co-ordinate action of P2Y and P2X7 receptors are critical for controlling LPS responses in vivo and that agents directed against both receptor classes may provide the greatest therapeutic advantage.
During infection or inflammation, high concentrations of extracellular nucleotides are released into the inflammatory microenvironment, supplying a source of ligand for purinergic receptors that are present on many immune cell types. The P2X 7 receptor, a member of the P2X purinergic receptor family of ATP-gated ion channels, is thought to play an important role in monocyte/macrophage activation. One factor that can powerfully activate macrophages is bacterial lipopolysaccharide (endotoxin, LPS) and although the mechanisms involved in this process are not well understood, it is clear that LPS activation of macrophages is central to the development of septic shock in response to Gram-negative bacteria. Several lines of evidence have demonstrated strong modulatory effects of adenine nucleotides on the events associated with LPS stimulation of macrophages. Further, because the signal transduction cascades initiated in macrophages upon LPS exposure are similar to those resulting from P2X 7 receptor stimulation, and because antagonism of the P2X 7 receptor can attenuate LPS-stimulated signaling events and mediator release, the P2X 7 receptor has been implicated in the control of macrophage responses to LPS. In addition, our laboratory has identified a consensus LPS-binding motif at the extreme carboxyl terminus of the P2X 7 receptor, further supporting the potential for a direct interaction between LPS and this purinergic receptor. In this review, we discuss potential regulatory domains and structural features of the P2X 7 receptor and outline some of the signal transduction pathways activated by P2X 7 receptor agonists. Moreover, we present evidence supporting a critical role for the P2X 7 receptor in modulating or mediating some of the biological effects of LPS in macrophages. Drug Dev. Res. 53:91-104, 2001.
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