Eduardo R. Lazarowski

Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance

Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable. This is thought to be due to the release of ‘find-me’ signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages and dendritic cells, leading to the prompt clearance of the dying cells. However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to that of apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or the expression of ectopic CD39) abrogated the ability of apoptotic cell supernatants to recruit monocytes in vitro and in vivo. We then identified the ATP/UTP receptor P2Y2 as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y2-null mice. The relevance of nucleotides in apoptotic cell clearance in vivo was revealed by two approaches. First, in a murine air-pouch model, apoptotic cell supernatants induced a threefold greater recruitment of monocytes and macrophages than supernatants from healthy cells did; this recruitment was abolished by depletion of nucleotides and was significantly decreased in P2Y2-/- (also known as P2ry2-/-) mice. Second, clearance of apoptotic thymocytes was significantly impaired by either depletion of nucleotides or interference with P2Y receptor function (by pharmacological inhibition or in P2Y2-/- mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y2-dependent recruitment of phagocytes, and provide evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo.

Pannexin 1 channels mediate /`find-me/' signal release and membrane permeability during apoptosis

Apoptotic cells release ‘find-me’ signals at the earliest stages of death to recruit phagocytes. The nucleotides ATP and UTP represent one class of find-me signals, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 overexpression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the ‘selective’ plasma membrane permeability of early apoptotic cells to specific dyes. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.

Constitutive Release of ATP and Evidence for Major Contribution of Ecto-nucleotide Pyrophosphatase and Nucleoside Diphosphokinase to Extracellular Nucleotide Concentrations

Nucleotides are important extracellular signaling molecules. At least five mammalian P2Y receptors exist that are specifically activated by ATP, UTP, ADP, or UDP. Although the existence of ectoenzymes that metabolize extracellular nucleotides is well established, the relative flux of ATP and UTP through their extracellular metabolic products remains undefined. Therefore, we have studied the kinetics of accumulation and metabolism of endogenous ATP in the extracellular medium of four different cell lines. ATP concentrations reached a maximum immediately after change of medium and decreased thereafter with a single exponential decay (t½;1 ∼;230–40 min). ATP levels did not fall to zero but attained a base-line concentration that was independent of the medium volume and of the initial ATP concentration. Although the base-line concentration of ATP remained stable for up to 12 h, [γ-32P]ATP added to resting cells as a radiotracer was completely degraded within 120 min, indicating that steady state reflected a basal rate of ATP release balanced by ATP hydrolysis (20–200 fmol × min− 1 × cell− 6). High performance liquid chromatography analysis revealed that the γ-phosphate of ATP was rapidly, although transiently, transferred during steady state to species subsequently identified as UTP and GTP, indicating the existence of both ecto-nucleoside diphosphokinase activity and the accumulation of endogenous UDP and GDP. Conversely, addition of [γ-32P]UTP to resting cells resulted in transient formation of [γ-32P]ATP, indicating phosphorylation of endogenous ADP by nucleoside diphosphokinase. The final32P-products of [γ-32P]ATP metabolism were [32P]orthophosphoric acid and a 32P-labeled species that was further purified and identified as [32P]inorganic pyrophosphate. In C6 cells, the formation of [32P]pyrophosphate from [γ-32P]ATP at steady state exceeded by 3-fold that of [32P]orthophosphate. These results illustrate for the first time a constitutive release of ATP and other nucleotides and reveal the existence of a complex extracellular metabolic pathway for released nucleotides. In addition to the existence of an ecto-ATPase activity, our results suggest a major scavenger role of ecto-ATP pyrophosphatase and a transphosphorylating activity of nucleoside diphosphokinase.

Direct Demonstration of Mechanically Induced Release of Cellular UTP and Its Implication for Uridine Nucleotide Receptor Activation

ATP is released from most cell types and functions as an extracellular signaling molecule through activation of members of the two large families of P2X and P2Y receptors. Although three mammalian P2Y receptors have been cloned that are selectively activated by uridine nucleotides, direct demonstration of the release of cellular UTP has not been reported. Pharmacological studies of the P2Y4 receptor expressed in 1321N1 human astrocytoma cells indicated that this receptor is activated by UTP but not by ATP. Mechanical stimulation of 1321N1 cells also resulted in release of a molecule that markedly activated the expressed P2Y4 receptor. This nucleotide was shown to be UTP by two means. First, high performance liquid chromatography analysis of the medium from [33P]H3PO4-loaded 1321N1 cells illustrated that mechanical stimulation resulted in a large increase in a radioactive species that co-eluted with authentic UTP. This species was degraded by incubation with the nonspecific pyrophosphohydrolase apyrase or with hexokinase and was specifically lost by incubation with the UTP-specific enzyme UDP-glucose pyrophosphorylase. Second, a sensitive assay that quantitates UTP mass at low nanomolar concentrations was devised based on the nucleotide specificity of UDP-glucose pyrophosphorylase. Using this assay, mechanical stimulation of 1321N1 cells was shown to result in an increase of medium UTP levels from 2.6 to 36.4 pmol/106cells within 2 min. This increase was paralleled by a similar augmentation of extracellular ATP levels. A calcein-based fluorescence quenching method was utilized to confirm that none of the increases in medium nucleotide levels could be accounted for by cell lysis. Taken together, these results directly demonstrate the mechanically induced release of UTP and illustrate the efficient coupling of this release to activation of P2Y4 receptors.

Pharmacological selectivity of the cloned human P2U-purinoceptor : potent activation by diadenosine tetraphosphate

1 The human P2U‐purinoceptor was stably expressed in 1321N1 human astrocytoma cells and the pharmacological selectivity of the expressed receptor was studied by measurement of inositol lipid hydrolysis. 2 High basal levels of inositol phosphates occurred in P2U‐purinoceptor‐expressing cells. This phenomenon was shown to be due to release of large amounts of ATP from 1321N1 cells, and could be circumvented by adoption of an assay protocol that did not involve medium changes. 3 UTP, ATP and ATPγS were full and potent agonists for activation of phospholipase C with EC50 values of 140 nM, 230 nM, and 1.72 μM, respectively. 5BrUTP, 2C1ATP and 8BrATP were also full agonists although less potent than their natural congeners. Little or no effect was observed with the selective P2Y‐, P2X‐, and P2T‐purinoceptor agonists, 2MeSATP, α,β‐MeATP, and 2MeSADP, respectively. 4 Diadenosine tetraphosphate, Ap4A, was a surprisingly potent agonist at the expressed P2U‐purinoceptor with an EC50 (720 nM) in the range of the most potent P2U‐purinoceptor agonists. AP4A may be a physiologically important activator of P2U‐purinoceptors.

PGC-1a Rescues Huntington's Disease Proteotoxicity by Preventing Oxidative Stress and Promoting TFEB Function

PGC-1α rescues Huntington’s disease neurodegeneration by reducing reactive oxygen species and inducing a master regulator of autophagy. From PGC-1α to TFEB: Mileposts in Huntington’s Disease Huntington’s disease (HD) is an untreatable neurodegenerative disease caused by the production of huntingtin protein containing too many polyglutamine repeats. Patients with HD develop a progressive movement disorder and cognitive decline for which no suitable therapy is currently available. HD pathogenesis stems from mitochondrial dysfunction and altered transcription of nuclear genes, which has been linked to impaired action of the transcription factor PPARγ co-activator 1α (PGC-1α). In a new study, Tsunemi et al. tested whether increased PGC-1α function could ameliorate neuronal loss and some of the neurological symptoms of HD by crossing a mouse in which PGC-1α can be inducibly overexpressed with a transgenic mouse model of HD. The authors found that not only does PGC-1α overexpression improve neurological function in these mice, but it also virtually eradicates aggregates of mutant huntingtin protein in the brains of the HD mice. When they analyzed the reason for this genetic rescue in cultured cells and in the HD mice, they discovered that increased PGC-1α expression promoted mitochondrial function by boosting oxidative phosphorylation and reducing oxidative stress by turning on the expression of genes that quench the damaging effects of reactive oxygen species. However, it turns out that the key to PGC-1α’s ability to induce clearance of mutant huntingtin protein aggregates is its capacity to switch on the expression of TFEB, a master regulatory transcription factor that activates genes in the autophagy-lysosome pathway of protein turnover. Regulation of TFEB by PGC-1α underscores the importance of maintaining mitochondrial quality control under conditions of accelerated mitochondrial biogenesis and increased ATP generation. As altered energy production and impaired protein quality control are key features of other neurodegenerative diseases like Parkinson’s disease, the new findings suggest that PGC-1α and TFEB may be useful therapeutic targets not only for HD but for other neurodegenerative disorders as well. Huntington’s disease (HD) is caused by CAG repeat expansions in the (huntingtin htt) gene, yielding proteins containing polyglutamine repeats that become misfolded and resist degradation. Previous studies demonstrated that mutant htt interferes with transcriptional programs coordinated by the peroxisome proliferator–activated receptor γ (PPARγ) coactivator 1α (PGC-1α), a regulator of mitochondrial biogenesis and oxidative stress. We tested whether restoration of PGC-1α could ameliorate the symptoms of HD in a mouse model. We found that PGC-1α induction virtually eliminated htt protein aggregation and ameliorated HD neurodegeneration in part by attenuating oxidative stress. PGC-1α promoted htt turnover and the elimination of protein aggregates by activating transcription factor EB (TFEB), a master regulator of the autophagy-lysosome pathway. TFEB alone was capable of reducing htt aggregation and neurotoxicity, placing PGC-1α upstream of TFEB and identifying these two molecules as important therapeutic targets in HD and potentially other neurodegenerative disorders caused by protein misfolding.

Compartmentalized autocrine signaling to cystic fibrosis transmembrane conductance regulator at the apical membrane of airway epithelial cells

Physical stimulation of airway surfaces evokes liquid secretion, but the events that mediate this vital protective function are not understood. When cystic fibrosis transmembrane conductance regulator (CFTR) channel activity was used as a functional readout, we found signaling elements compartmentalized at both extracellular and intracellular surfaces of the apical cell membrane that activate apical Cl− conductance in Calu-3 cells. At the outer surface, ATP was released by physical stimuli, locally converted to adenosine, and sensed by A2B adenosine receptors. These receptors couple to G proteins, adenylyl cyclase, and protein kinase A, at the intracellular face of the apical membrane to activate colocalized CFTR. Thus, airways have evolved highly efficient mechanisms to “flush” noxious stimuli from airway surfaces by selective activation of apical membrane signal transduction and effector systems.

Effect of Loss of P2Y2 Receptor Gene Expression on Nucleotide Regulation of Murine Epithelial Cl−Transport

Extracellular nucleotides are believed to be important regulators of ion transport in epithelial tissues as a result of their ability to activate cell surface receptors. Although numerous receptors that bind nucleotides have been identified, the complexity of this receptor family, combined with the lack of pharmacological agents specific for these receptors, has made the assignment of particular receptors and ligands to physiological responses difficult. Because ATP and UTP appear equipotent and equieffective in regulating ion transport in many epithelia, we tested the hypothesis that the P2Y2 receptor (P2Y2-R) subtype mediates these responses in mouse epithelia, with gene targeting techniques. Mice with the P2Y 2-R locus targeted and inactivated (P2Y2-R(−/−)) were generated, airways (trachea), gallbladder, and intestines (jejunum) excised, and Cl− secretory responses to luminal nucleotide additions measured in Ussing chambers. Comparison of P2Y2-R(+/+) with P2Y2-R(−/−) mice revealed that P2Y2-R mediated most (>85–95%) nucleotide-stimulated Cl−secretion in trachea, about 50% of nucleotide responses in the gallbladder, and none of the responses in the jejunum. Dose-effect relationships for nucleotides in tissues from P2Y2-R(−/−) mice suggest that the P2Y6-R regulates ion transport in gallbladder and to a lesser extent trachea, whereas P2Y4and/or unidentified receptor(s) regulate ion transport in jejunum. We conclude that the P2Y2 receptor is the dominant P2Y purinoceptor that regulates airway epithelial ion transport, whereas other P2Y receptor subtypes are relatively more important in other nonrespiratory epithelia.

Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase

Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5′-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H2O2 was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1–312), we found that mutation of cysteine 299 to alanine diminished the ability of H2O2 to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H2O2 on kinase activity. Similar to the results obtained with H2O2-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H2O2 are increased. These results demonstrate that physiologically relevant concentrations of H2O2 can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status.

Physiological Regulation of ATP Release at the Apical Surface of Human Airway Epithelia

Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin (∼7 μm) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1-10 nm. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of ∼250 fmol/min/cm2, reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached ∼1 μm independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intracellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation.