The Pannexin 1 Channel Activates the Inflammasome in Neurons and Astrocytes
2009; Elsevier BV; Volume: 284; Issue: 27 Linguagem: Inglês
10.1074/jbc.m109.004804
ISSN1083-351X
AutoresWilliam R. Silverman, Juan Pablo de Rivero Vaccari, Silviu Locovei, Feng Qiu, Steven K. Carlsson, Eliana Scemes, Robert W. Keane, Gerhard Dahl,
Tópico(s)Connexins and lens biology
ResumoThe inflammasome is a multiprotein complex involved in innate immunity. Activation of the inflammasome causes the processing and release of the cytokines interleukins 1β and 18. In primary macrophages, potassium ion flux and the membrane channel pannexin 1 have been suggested to play roles in inflammasome activation. However, the molecular mechanism(s) governing inflammasome signaling remains poorly defined, and it is undetermined whether these mechanisms apply to the central nervous system. Here we show that high extracellular potassium opens pannexin channels leading to caspase-1 activation in primary neurons and astrocytes. The effect of K+ on pannexin 1 channels was independent of membrane potential, suggesting that stimulation of inflammasome signaling was mediated by an allosteric effect. The activation of the inflammasome by K+ was inhibited by the pannexin 1 channel blocker probenecid, supporting a role of pannexin 1 in inflammasome activation. Co-immunoprecipitation of neuronal lysates indicates that pannexin 1 associates with components of the multiprotein inflammasome complex, including the P2X7 receptor and caspase-1. Moreover antibody neutralization of the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) blocked ATP-induced cell death in oocytes co-expressing P2X7 receptor and pannexin 1. Thus, in contrast to macrophages and monocytes in which low intracellular K+ has been suggested to trigger inflammasome activation, in neural cells, high extracellular K+ activates caspase-1 probably through pannexin 1. The inflammasome is a multiprotein complex involved in innate immunity. Activation of the inflammasome causes the processing and release of the cytokines interleukins 1β and 18. In primary macrophages, potassium ion flux and the membrane channel pannexin 1 have been suggested to play roles in inflammasome activation. However, the molecular mechanism(s) governing inflammasome signaling remains poorly defined, and it is undetermined whether these mechanisms apply to the central nervous system. Here we show that high extracellular potassium opens pannexin channels leading to caspase-1 activation in primary neurons and astrocytes. The effect of K+ on pannexin 1 channels was independent of membrane potential, suggesting that stimulation of inflammasome signaling was mediated by an allosteric effect. The activation of the inflammasome by K+ was inhibited by the pannexin 1 channel blocker probenecid, supporting a role of pannexin 1 in inflammasome activation. Co-immunoprecipitation of neuronal lysates indicates that pannexin 1 associates with components of the multiprotein inflammasome complex, including the P2X7 receptor and caspase-1. Moreover antibody neutralization of the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) blocked ATP-induced cell death in oocytes co-expressing P2X7 receptor and pannexin 1. Thus, in contrast to macrophages and monocytes in which low intracellular K+ has been suggested to trigger inflammasome activation, in neural cells, high extracellular K+ activates caspase-1 probably through pannexin 1. Pannexin 1 is a vertebrate ortholog of the invertebrate innexin gap junction proteins (1.Panchin Y. Kelmanson I. Matz M. Lukyanov K. Usman N. Lukyanov S. Curr. Biol. 2000; 10: R473-474Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar), but it does not appear to form functional gap junctions in vivo. Instead pannexin 1 acts as a membrane channel that carries ions and signaling molecules between the cytoplasm and the extracellular space (2.Dahl G. Locovei S. IUBMB Life. 2006; 58: 409-419Crossref PubMed Scopus (149) Google Scholar, 3.Huang Y. Grinspan J.B. Abrams C.K. Scherer S.S. Glia. 2007; 55: 46-56Crossref PubMed Scopus (150) Google Scholar). As such, it is a candidate ATP release channel in various cell types, including erythrocytes, astrocytes, bronchial epithelial cells, and taste cells. Various functional roles have been ascribed to pannexin 1 including local vascular perfusion control and propagation of intercellular calcium waves (4.Locovei S. Bao L. Dahl G. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 7655-7659Crossref PubMed Scopus (425) Google Scholar, 5.Iglesias R. Locovei S. Roque A. Alberto A.P. Dahl G. Spray D.C. Scemes E. Am. J. Physiol. Cell Physiol. 2008; 295: C752-760Crossref PubMed Scopus (281) Google Scholar, 6.Scemes E. Suadicani S.O. Dahl G. Spray D.C. Neuron Glia Biol. 2007; 3: 199-208Crossref PubMed Scopus (183) Google Scholar). Recently pannexin 1 was also shown to form the large pore of the P2X7 purinergic receptor (7.Pelegrin P. Surprenant A. EMBO J. 2006; 25: 5071-5082Crossref PubMed Scopus (1147) Google Scholar, 8.Locovei S. Scemes E. Qiu F. Spray D.C. Dahl G. FEBS Lett. 2007; 581: 483-488Crossref PubMed Scopus (361) Google Scholar). P2X7 plays a major role in inflammation, and its activation by extracellular ATP results in release of interleukin (IL) 2The abbreviations used are: ILinterleukinNLRNOD (nucleotide-binding oligomerization domain)-like receptorASCapoptosis-associated specklike protein containing a CARD (caspase recruitment domain)CNScentral nervous systemXIAPX-linked inhibitor of apoptosis proteinPBSphosphate-buffered salineshshort hairpin. -1β from macrophages, probably involving pannexin 1 as a signaling molecule (7.Pelegrin P. Surprenant A. EMBO J. 2006; 25: 5071-5082Crossref PubMed Scopus (1147) Google Scholar). interleukin NOD (nucleotide-binding oligomerization domain)-like receptor apoptosis-associated specklike protein containing a CARD (caspase recruitment domain) central nervous system X-linked inhibitor of apoptosis protein phosphate-buffered saline short hairpin. IL-1β production and maturation are tightly regulated by caspase-1 incorporated into large protein complexes termed inflammasomes (9.Martinon F. Burns K. Tschopp J. Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (4307) Google Scholar, 10.Martinon F. Tschopp J. Cell Death Differ. 2007; 14: 10-22Crossref PubMed Scopus (670) Google Scholar, 11.Ogura Y. Sutterwala F.S. Flavell R.A. Cell. 2006; 126: 659-662Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). The molecular composition of the inflammasome depends on the identity of the NOD-like receptor (NLR) family member serving as a scaffold protein in the complex (12.Ting J.P. Willingham S.B. Bergstralh D.T. Nat. Rev. Immunol. 2008; 8: 372-379Crossref PubMed Scopus (289) Google Scholar). The members of the cytosolic NLR family appear to recognize conserved microbial and viral components termed pathogen-associated molecular patterns in intracellular compartments (13.Carneiro L.A. Magalhaes J.G. Tattoli I. Philpott D.J. Travassos L.H. J. Pathol. 2008; 214: 136-148Crossref PubMed Scopus (154) Google Scholar). The bipartite adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) bridges the interaction between NLR proteins and inflammatory caspases and plays a central role in the assembly of inflammasomes and the activation of caspase-1 in response to a broad range of pathogen-associated molecular patterns and intracellular pathogens (14.Taniguchi S. Sagara J. Semin. Immunopathol. 2007; 29: 231-238Crossref PubMed Scopus (67) Google Scholar). In addition, the inflammasome can be activated by danger-associated molecular patterns, molecules endogenous to the organism that signal stress or injury, including extracellular ATP acting at ionotropic P2X7 receptors, fibronectin, or monosodium urate crystals (15.Matzinger P. Ann. N.Y. Acad. Sci. 2002; 961: 341-342Crossref PubMed Scopus (225) Google Scholar, 16.Matzinger P. Nat. Immunol. 2007; 8: 11-13Crossref PubMed Scopus (407) Google Scholar). Moreover it has been suggested that a rapid K+ efflux through ATP-activated P2X7 receptors induces inflammasome assembly (17.Perregaux D. Gabel C.A. J. Biol. Chem. 1994; 269: 15195-15203Abstract Full Text PDF PubMed Google Scholar, 18.Walev I. Reske K. Palmer M. Valeva A. Bhakdi S. EMBO J. 1995; 14: 1607-1614Crossref PubMed Scopus (238) Google Scholar, 19.Colomar A. Marty V. Médina C. Combe C. Parnet P. Amédée T. J. Biol. Chem. 2003; 278: 30732-30740Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 20.Pétrilli V. Papin S. Dostert C. Mayor A. Martinon F. Tschopp J. Cell Death Differ. 2007; 14: 1583-1589Crossref PubMed Scopus (1091) Google Scholar). Despite the recent advances in the understanding of accessory proteins required for full activation of caspase-1, little is known about the signaling pathways that trigger inflammasome activation, particularly in the central nervous system (CNS). Recently we reported that spinal cord neurons contain the NLRP1/NALP1 inflammasome consisting of NLRP1, ASC, caspase-1, caspase-11, and the X-linked inhibitor of apoptosis protein (XIAP) and that spinal cord injury induces rapid activation of the inflammasome, causing processing and secretion of IL-1β and IL-18. Moreover antibody neutralization of ASC reduces caspase-1 activation and IL-1 cytokine processing, leading to significant tissue sparing and functional improvement (21.de Rivero Vaccari J.P. Lotocki G. Marcillo A.E. Dietrich W.D. Keane R.W. J. Neurosci. 2008; 28: 3404-3414Crossref PubMed Scopus (277) Google Scholar). In this study, we focused on signaling events coupling pannexin 1 and P2X7 receptors to rapid caspase-1 activation in primary neurons and astrocytes. We provide compelling evidence that high extracellular K+ opens the pannexin 1 channel and activates inflammasomes in neurons and astrocytes, but not THP-1 cells, thus leading to caspase-1 activation. This signaling pathway in neurons is mediated through protein interactions between pannexin 1 and inflammasome proteins. We also provide evidence that ATP acting on P2X7 induces rapid cell death and that antibody neutralization of ASC blocks ATP-induced cell death. Thus, contrary to the widely accepted view in macrophages and monocytes that low intracellular K+ triggers inflammasome activation, high extracellular K+ surrounding cells such as neurons and astrocytes opens pannexin 1 channels and induces processing of caspase-1. Rabbit anti-Rattus norvegicus ASC and NLRP1 antisera were prepared by Bethyl Laboratories as described previously (21.de Rivero Vaccari J.P. Lotocki G. Marcillo A.E. Dietrich W.D. Keane R.W. J. Neurosci. 2008; 28: 3404-3414Crossref PubMed Scopus (277) Google Scholar). Chicken anti-pannexin 1 (4515) has been characterized (4.Locovei S. Bao L. Dahl G. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 7655-7659Crossref PubMed Scopus (425) Google Scholar), and antibody affinity-purified on a matrix with the cognate peptide (prepared by Aves Labs Inc.) was used. Other antibodies were purchased from commercial sources and included anti-NLRP1 (Abcam), anti-IL-1β (Cell Signaling Technology Inc.), anti-caspase-1 (Upstate); anti-caspase-1 (Santa Cruz Biotechnology, Inc.), anti-caspase-11 (Alexis Biochemicals), anti-caspase-11 (Santa Cruz Biotechnology, Inc.), anti-XIAP (BD Transduction Laboratories), anti-caspase-3 (Santa Cruz Biotechnology, Inc.), and anti-P2X7 receptor (Calbiochem). Neuronal cultures were prepared from embryonic day 16–17 rat cortices as described previously (22.Tedeschi B. Barrett J.N. Keane R.W. J. Cell Biol. 1986; 102: 2244-2253Crossref PubMed Scopus (128) Google Scholar, 23.Keane R.W. Tallent M.W. Podack E.R. Transplantation. 1992; 54: 520-526Crossref PubMed Scopus (25) Google Scholar). Cortical tissue was disrupted into a cell suspension by gentle trituration and seeded in 60-mm dishes at a density of 2 × 106 cells/dish. Neurons were grown on poly-l-lysine-coated tissue culture dishes in N5 medium that contained 5% serum fraction (24.Kawamoto J.C. Barrett J.N. Brain Res. 1986; 384: 84-93Crossref PubMed Scopus (155) Google Scholar). Neurons were maintained for 12 days, and the neuronal nature of the majority of cells (95%) was confirmed electrophysiologically and immunohistochemically (22.Tedeschi B. Barrett J.N. Keane R.W. J. Cell Biol. 1986; 102: 2244-2253Crossref PubMed Scopus (128) Google Scholar). Primary astrocytes were obtained from neonatal rat cerebral cortices as described previously (22.Tedeschi B. Barrett J.N. Keane R.W. J. Cell Biol. 1986; 102: 2244-2253Crossref PubMed Scopus (128) Google Scholar). The cells were maintained for 3 weeks in Dulbecco's modified Eagle's medium supplemented with 10% horse serum. At least 99% of the cell populations were astrocytes as determined by staining with cell-specific markers (22.Tedeschi B. Barrett J.N. Keane R.W. J. Cell Biol. 1986; 102: 2244-2253Crossref PubMed Scopus (128) Google Scholar). The human monocytic cell line THP-1 (American Type Culture Collection) was maintained in RPMI 1640 medium supplemented with 0.05 mm 2-mecaptoethanol and 10% fetal bovine serum. Cells were pretreated with 1 mm probenecid (Alfa Aesar) for 10 min. The medium was removed and replaced with medium containing probenecid and 130 mm KCl for 30 min, 1 h, and 2 h, whereas controls received probenecid alone. Cells were washed once in ice-cold PBS and lysed as described previously (25.Keane R.W. Srinivasan A. Foster L.M. Testa M.P. Ord T. Nonner D. Wang H.G. Reed J.C. Bredesen D.E. Kayalar C. J. Neurosci. Res. 1997; 48: 168-180Crossref PubMed Scopus (147) Google Scholar) and prepared for immunoblot analysis. Pannexin 1 was knocked down using short hairpin (sh)RNA inserted into a retroviral silencing plasmid (pRS) purchased from Origene. One microgram of Panx1 shRNA expression plasmids containing puromycin resistance was transfected into the human 1321N1 astrocytoma cells plated in 35-mm dishes using Lipofectamine 2000 reagent. After overnight incubation, transfection reagents were removed, and cells were transferred to 100-mm dishes containing selection medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1 μg/ml puromycin). After 2–3 weeks in selection medium, clones were tested for appropriate Panx1 knockdown (18.Walev I. Reske K. Palmer M. Valeva A. Bhakdi S. EMBO J. 1995; 14: 1607-1614Crossref PubMed Scopus (238) Google Scholar) using Western blot analysis. The selected Panx1-KD 1321N1 cells were maintained in selection medium in a humidified chamber (100% humidity, 95% air, 5% CO2, 37 °C). Primary cell cultures were lysed in lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm pyrophosphate, 1 mm β-glycerophosphate) with protease inhibitor mixture (Sigma-Aldrich). For immunoblot analysis of oocytes, each lane contained extracts of three oocytes that were pooled and lysed in 50 μl of oocyte Ringer's solution (OR2) by repeated passage through the end of a 1-ml disposable pipette tip. Cells were spun at 12,000 × g for 3 min, and samples were taken from the supernatant, avoiding both the pellet and the lipids on the surface. Laemmli sample buffer was added, and proteins were resolved on 10–20% Tris-HCl Criterion precast gels (Bio-Rad), transferred to polyvinylidene difluoride membranes (Millipore), placed in blocking buffer (PBS, 0.1% Tween 20, 0.4% I-Block (Applied Biosystems)), and then incubated for 1 h with primary antibodies followed by appropriate secondary horseradish peroxidase (HRP)-linked antibodies (Cell Signaling Technology Inc.). Visualization of signal was enhanced by chemiluminescence using a Phototope-HRP detection kit (Cell Signaling Technology Inc.). To control for protein loading, immunoblots were stripped with Restore Western blot stripping buffer (26.Doucet J.P. Pierce G.N. Hertzberg E.L. Tuana B.S. J. Biol. Chem. 1992; 267: 16503-16508Abstract Full Text PDF PubMed Google Scholar) and blotted for β-tubulin using monoclonal anti-β-tubulin antibody (1:5000, BD Biosciences Pharmingen). Quantification of band density was performed using UN-SCAN-IT gelTM digitizing software (Silk Scientific, Inc.), and data were normalized to β-tubulin. To assess the protein composition and association of proteins in the inflammasome, primary neuronal cultures (2 × 106 cells) were lysed in 200 μl of lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm pyrophosphate, 1 mm β-glycerophosphate) with protease inhibitor mixture (Sigma-Aldrich). Approximately 200 μg of neuronal lysates were immunoprecipitated with anti-ASC or anti-pannexin 1 antibodies using TrueBlotTM anti-rabbit Ig or PrecipHen (Aves Labs Inc.) immunoprecipitation beads. Neuronal lysates were precleared by adding 50 μl of anti-rabbit TrueBlot beads to 200 μg of lysate in a microcentrifuge tube. The mixture was incubated for 1 h at 4 °C, and beads were pelleted by centrifugation at 12,000 × g for 30 s. The supernatant was recovered and immunoprecipitated with 5 μg of anti-ASC or anti-pannexin 1 and incubated at 4 °C overnight. Fifty microliters of anti-rabbit TrueBlot beads or anti-chicken PrecipHen beads were added to the mixture, incubated for 2 h, and then centrifuged at 12,000 × g for 30 s. The pelleted beads were washed five times in lysis buffer, resuspended in loading buffer, and heated at 95 °C for 3 min before analysis by immunoblotting using antibodies against ASC, NLRP1, capase-11 and caspase-1, caspase-3, XIAP, and pannexin 1. Oocytes were prepared as described previously (27.Dahl G. Stevenson B. Gallin W. Paul D. Cell-cell Interactions. A Practical Approach. IRL Press, Oxford1992: 143-165Google Scholar). Xenopus laevis oocytes were isolated by incubating small pieces of ovary in 2 mg/ml collagenase type I (Worthington) in Ca2+-free OR2 (82.5 mm NaCl, 2.5 mm KCl, 1.0 mm MgCl2, 1.0 mm CaCl2, 1.0 mm Na2HPO4, 5.0 mm HEPES (pH 7.5) with antibiotics (10,000 units/ml penicillin and 10 mg/ml streptomycin) and stirring at one turn/s for 3 h at room temperature. After thorough washing with regular OR2, oocytes devoid of follicle cells and having a uniform pigmentation were selected and stored in OR2 at 18 °C. RNA for mouse pannexin 1 was prepared using the mMessage mMachine in vitro transcription kit (Ambion). Oocytes were injected with 20–40 nl of cRNA (1 μg/μl) and incubated for 18–48 h at 18 °C. RNA for human pannexin 1 and P2X7 was similarly prepared and injected. Oocytes were then incubated for 7–8 days prior to electrophysiological analysis at room temperature. Oocytes were tested using two-electrode voltage clamp (Model OC725C, Warner Instruments) under constant perfusion according to the protocols described in the figures. Oocytes expressing both human pannexin 1 and P2X7 receptor were tested 7–8 days after injection of RNA. Cells were clamped at −40 mV, and 5-s test pulses to −35 mV were applied. After base-line readings were taken in OR2, cells were exposed to 500 μm ATP for 4 min and then returned to OR2. After it was established that the ATP reliably resulted in cell death, remaining co-expressing cells were then injected with 60 nl of anti-ASC (diluted 1:10 in PBS) or 60 nl of control IgG (diluted 1:10 in PBS). After 1 h, the reinjected cells were exposed to 500 μm ATP and tested as before. Parental and Panx1-KD 1321N1 human astrocytoma cells were exposed for 30 min to control solution (145 mm NaCl, 5 mm KCl, 1.4 mm CaCl2, 1.0 mm MgCl2, 10 mm HEPES, pH 7.4) and to a depolarizing solution (60 mm NaCl, 50 mm KCl, 1.4 mm CaCl2, 1.0 mm MgCl2, 10 mm HEPES, pH 7.4) containing 10 μm YoPro1. After treatment, cells were washed with control solution and then fixed with p-formaldehyde, and cover slips were mounted using Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole. YoPro fluorescence intensity was measured from the whole field of view of high K+ (50 mm)-treated cells and normalized to that obtained under control (5 mm K+) conditions. Images were acquired using MetaFluor software (Universal Imaging Corp.) and a digital camera (Photometrics HQ2) attached to a Nikon inverted TE-2000E microscope equipped with a 10× objective and fluorescein isothiocyanate and 4′,6-diamidino-2-phenylindole filter sets. Pannexin 1 is a channel that releases ATP and other ions from the cell. Accordingly an increase of extracellular K+ concentration causes ATP release from Xenopus oocytes expressing pannexin 1 (2.Dahl G. Locovei S. IUBMB Life. 2006; 58: 409-419Crossref PubMed Scopus (149) Google Scholar, 28.Bao L. Locovei S. Dahl G. FEBS Lett. 2004; 572: 65-68Crossref PubMed Scopus (637) Google Scholar, 29.Silverman W. Locovei S. Dahl G. Am. J. Physiol. Cell Physiol. 2008; 295: C761-767Crossref PubMed Scopus (314) Google Scholar). However, electrophysiological data suggest that pannexin 1 opens only when depolarized to positive membrane potentials. To investigate this apparent discrepancy, we applied 130 mm K+ to pannexin 1 channels in voltage-clamped oocytes held at potentials at which the channel is normally closed. When high extracellular K+ was applied, currents in response to a 10-mV test pulse from a holding potential of −50 mV increased in a reproducible and reversible fashion (Fig. 1a and supplementalFig. 1A). The K+-induced current was attenuated by carbenoxolone (Fig. 1a) and by probenecid (supplementalFig. 2). In contrast, application of extracellular K+ to noninjected oocytes increased conductance only moderately (Fig. 1b). To quantify this increase, we measured the conductance of oocytes to this test pulse and found that 130 mm K+ increased conductance substantially in pannexin 1-expressing oocytes (Fig. 1c). Application of 130 mm K+ also increased the holding current needed to maintain cells at −50 mV compared with noninjected oocytes (Fig. 1d). The effect of K+ was dose-dependent. The lowest potassium concentration required for detectable activation of pannexin 1 currents was 20 mm (supplementalFig. 1B). Replacement of Na+ with choline+ did not activate pannexin (not shown), indicating that the observed effects are due to K+ and not the removal of Na+. Potassium chloride was as effective as potassium gluconate to activate pannexin 1 channels (not shown).FIGURE 2Voltage dependence of activation of pannexin 1 channels by high extracellular K+. A, currents from oocytes held in OR2 or perfused with 140 mm potassium gluconate (KGlu). Cells were held at −100 mV and subjected to a voltage ramp lasting 1 min from −100 mV to + 100 mV as indicated below the current traces. In OR2, cells expressing mPanx1 showed much larger current than noninjected cells, and addition of carbenoxolone (CBX) (100 μm) reduced mPanx1 currents to noninjected levels. Carbenoxolone had no effect on noninjected cells. Addition of potassium gluconate to mPanx1-expressing cells resulted in inward currents at −100 mV and increased outward currents at positive potentials. Carbenoxolone dramatically reduced both the inward and outward currents. B, quantification of currents recorded at −100, −50, 0, and +50 mV. Data are mean ± S.E.; n = 4–5. Where not visible, error bars are smaller than symbols.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The activation of pannexin 1 currents by high extracellular K+ was observed over a wide range of voltages (Fig. 2) with depolarization facilitating the process. Only at high positive potentials was the effect of high K+ minimal, probably because of exhaustion of recruitable channels. Taken together, these data support the hypothesis that high extracellular K+ opens pannexin 1 channels at resting potentials at which the channel is normally closed. To test whether K+ also stimulates pannexin 1-mediated dye uptake, we used the astrocytoma cell line 1321N1. Thirty-minute exposure of 1321N1 cells to a depolarizing solution (50 mm K+) caused a significant influx of YoPro1 into cells compared with those bathed in normal (5 mm K+) solution (control, 0.99 ± 0.012-fold; high K+, 1.24 ± 0.031-fold; n = 6 fields from three experiments) (Fig. 3). High K+-induced dye uptake was significantly attenuated (0.99 ± 0.017-fold, n = 6 fields from three experiments) when Panx1 was knocked down with Panx1 shRNA (Fig. 3). Distinguishing the functions of pannexin 1 channels from connexin channels has been hampered by the lack of specific inhibitors able to discriminate between them. Despite the lack of any sequence homology between pannexins and connexins, known inhibitors of connexins also inhibit pannexin channels. Carbenoxolone demonstrates some specificity as its dose dependence for inhibition of pannexin and connexins differs by a factor of ∼3 (30.Bruzzone R. Barbe M.T. Jakob N.J. Monyer H. J. Neurochem. 2005; 92: 1033-1043Crossref PubMed Scopus (384) Google Scholar). We have reported that a more specific inhibitor of pannexin 1 is probenecid, a common drug used to treat gout and gouty arthritis (29.Silverman W. Locovei S. Dahl G. Am. J. Physiol. Cell Physiol. 2008; 295: C761-767Crossref PubMed Scopus (314) Google Scholar). Application of probenecid rapidly inhibited voltage-induced pannexin 1 currents in oocytes as did carbenoxolone (Fig. 4). We also found that probenecid significantly reduced the currents in response to application of 130 mm extracellular K+ (supplementalFig. 2), providing additional evidence that pannexin 1 is responsible for the currents in response to the extracellular K+ application. Neurons in culture express the NLRP1 inflammasome (21.de Rivero Vaccari J.P. Lotocki G. Marcillo A.E. Dietrich W.D. Keane R.W. J. Neurosci. 2008; 28: 3404-3414Crossref PubMed Scopus (277) Google Scholar). To test whether high extracellular K+ activates inflammasomes and caspase-1, we treated rat cortical neurons and astrocytes and human THP-1 cells grown in culture with 130 mm KCl for 30 min, 1 h, and 2 h and assayed protein lysates for caspase-1 activation. As shown in Fig. 5, neurons and astrocytes exposed to high extracellular K+ showed an increase in caspase-1 activation when compared with untreated controls. As shown by others (20.Pétrilli V. Papin S. Dostert C. Mayor A. Martinon F. Tschopp J. Cell Death Differ. 2007; 14: 1583-1589Crossref PubMed Scopus (1091) Google Scholar), human THP-1 cells were refractory to high extracellular K+ exposure and therefore served as control. Pretreatment with the pannexin channel blocker probenecid (1 mm) prior to high K+ treatment suppressed caspase-1 induction in neurons and astrocytes (Fig. 5). Thus, inflammasome activation and caspase-1 processing induced by high extracellular K+ are inhibited by blocking the pannexin 1 channel, suggesting a role for pannexin 1 in the activation of the inflammasome in neurons and astrocytes but not THP-1 cells. Stimulation of the inflammasome in neurons and astrocytes also led to the release of IL-1β (supplementalFig. 3). Curiously the supernatant contained more of the unprocessed than the mature form of IL-1β. The rapid increase in caspase-1 expression after stimulation is in agreement with observations made in CNS injury models (21.de Rivero Vaccari J.P. Lotocki G. Marcillo A.E. Dietrich W.D. Keane R.W. J. Neurosci. 2008; 28: 3404-3414Crossref PubMed Scopus (277) Google Scholar, 31.Pineau I. Lacroix S. J. Comp. Neurol. 2007; 500: 267-285Crossref PubMed Scopus (454) Google Scholar, 32.Abulafia D.P. de Rivero Vaccari J.P. Lozano J.D. Lotocki G. Keane R.W. Dietrich W.D. J. Cereb. Blood Flow Metab. 2009; 29: 534-544Crossref PubMed Scopus (264) Google Scholar). A similar rapid increase of caspase-1 is also observed in macrophages exposed to Gram-negative bacteria or other agents like pathogen-associated molecular patterns (9.Martinon F. Burns K. Tschopp J. Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (4307) Google Scholar, 33.Mariathasan S. Newton K. Monack D.M. Vucic D. French D.M. Lee W.P. Roose-Girma M. Erickson S. Dixit V.M. Nature. 2004; 430: 213-218Crossref PubMed Scopus (1428) Google Scholar). For an alternate test for a role of pannexin 1 in the activation of the inflammasome by K+ we used the 1321N1 cell line. The parental cell line exhibited an activation pattern of caspase-1 similar to that of primary astrocytes and neurons in response to stimulation with high K+. In contrast, caspase-1 in the pannexin 1 knockdown line was barely detectable and did not show increased activation (Fig. 6). The reduced expression of caspase-1 could be due to either transcriptional/translational effects or stability of the various components of the inflammasome multiprotein complex. It is conceivable that unassembled caspase-1 is degraded faster than the complexed protein. To characterize associations of inflammasome proteins in cortical neurons in culture, co-immunoprecipitations of neuronal lysates exposed to 130 mm KCl for 1 h were performed using anti-pannexin 1 antibody (Fig. 7, left). Anti-pannexin 1 immunoprecipitated NLRP1, ASC, caspase-1 and -11, XIAP, pannexin 1, and the P2X7 receptor. Caspase-1 was processed into its fragments (p26 and p13), and full-length XIAP was cleaved into a 30-kDa fragment. Thus, exposure to high extracellular K+ activates inflammasomes. Anti-pannexin 1 did not immunoprecipitate caspase-3, serving as a control. Caspase-3, however, was expressed in neurons as indicated by its presence in the lysate (supplementalFig. 4). Like caspase-1, caspase-3 levels increased with exposure to extracellular K+. In reciprocal co-immunoprecipitation experiments, anti-ASC immunoprecipitated NLRP1, ASC and caspase-1 and -11 as well as XIAP, pannexin 1, and the purinergic receptor P2X7 (Fig. 7, right). These findings demonstrate that the NLRP1 inflammasome in neurons associates with pannexin 1 and the P2X7 receptor. The co-immunoprecipitation pattern was similar in unstimulated and stimulated neurons, suggesting that the inflammasome complex is preassembled. This observation is consistent with findings that the NLRP1 inflammasome is preassembled in motor neurons of the spinal cord (21.de Rivero Vaccari J.P. Lotocki G. Mar
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