scholarly journals Novel Evidence That the Pannexin 1 Channel Is Involved in Adenosine Triphosphate (ATP) Release from Cells for Optimal Mobilization of Hematopoietic Stem Progenitor Cells, and the Pannexin 1 SNP 5 (Rs3020015) T/C Polymorphism Characterizes Poor Mobilizer Status in Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3248-3248
Author(s):  
Monika Cymer ◽  
Mateusz Adamiak ◽  
Marta Skoda ◽  
Marta Antonina Libura ◽  
Elzbieta Urbanowska ◽  
...  
Keyword(s):  
Adenosine Triphosphate ◽  
P2x7 Receptor ◽  
Conflicts Of Interest ◽  
Purinergic Receptor ◽  
Purinergic Signaling ◽  
Sterile Inflammation ◽  
Hematopoietic Stem ◽  
Pannexin 1 ◽  
Poor Mobilizer ◽  
Poor Mobilizers

Background . Pannexins have been shown to act predominantly as large transmembrane channels connecting the intracellular and extracellular space, allowing the passage of ions and small molecules, such as adenosine triphosphate (ATP), between these compartments. Pannexin 1 channels are involved in the release of ATP from cells and have been shown to be involved in the early stages of the innate immune response through an interaction with the P2X7 purinergic receptor. Recently, we provided evidence that activation of innate immunity in BM after administration of G-CSF produces "sterile inflammation" in the BM microenvironment and activates the pannexin channel to release ATP, which through binding to the P2X7 receptor leads to the mobilization of HSPCs (Leukemia 2018, 32:1920-1931). Corroborating our observation, it has been recently reported that the presence of the Gln460Arg SNP polymorphism within the P2X7 receptor gene in HSPCs, which is always co-inherited with Ala348Thr to form the gain-of-function haplotype 4, resulted in a significant increase in CD34+ HSPC mobilization (Leukemia 2018; 32:2724-2726). To shed more light on the novel concept that pannexin-1-released ATP, which interacts with P2X7, plays a crucial role in the egress of HSPCs from BM into peripheral blood (PB), we focused on the role of pannexin 1 in the mobilization process. Hypothesis. We hypothesized that pannexin 1 deficiency would negatively impact mobilization of HSPCs. Materials and Methods. First, we mobilized mice with G-CSF or AMD3100 in the presence of pannexin-1-blocking peptide. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. Next, we analyzed five types of polymorphisms in the human pannexin 1 gene (SNP1-Rs1138800 A/C, SNP2-Rs7928030 G/C, SNP3-Rs12294985 C/T, SNP4-Rs127933348 A/G, and SNP5-Rs3020015 T/C) and correlated them with good or poor mobilization status of the patients. Patients in our studies were mobilized in the Bone Marrow Transplant Unit, Warsaw Medical University with G-CSF using a standard mobilization protocol, and poor mobilizers were identified as patients who were not able to mobilize the required number of CD34+ cells according to standard criteria. In our studies DNA from patient blood samples was isolated and fragments of DNA amplified and subsequently sequenced. Our patients in the good- and poor-mobilizer groups were matched for age, sex, and basic disorders. Results. We found that mice with blocked pannexin 1 channels mobilized HSPCs significantly less efficiently. More importantly, our patient data revealed that ~60% of patients that turned out to be poor HSPC mobilizers (n=20) displayed the pannexin 1 polymorphism SNP5 (Rs3020015) T/C. This polymorphism was observed in only 1 out of 26 good-mobilizer patients. Conclusions. Our results further support an important role for ATP-mediated purinergic signaling in the mobilization of HSPCs. Moreover, the pannexin 1 SNP5Rs3020015 T/C polymorphism may serve as a diagnostic tool to identify poor mobilizers. This investigation shed more light on the molecular pathways involved in the egress of HSPCs from BM into PB and will help to design better mobilization protocols in the case of poor mobilizers. Disclosures No relevant conflicts of interest to declare.

scholarly journals Novel Evidence That Extracellular Nucleotides and Nucleosides Regulate the Expression of Heme Oxygenase 1 (HO-1) in Opposite Ways in Hematopoietic Stem/Progenitor Cells (HSPCs), Which Explains Why ATP Enhances Mobilization of HSPCs, While Its Metabolite Adenosine Inhibits This Process

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4528-4528
Author(s):  
Mateusz Adamiak ◽  
Kamila Bujko ◽  
Monika Plonka ◽  
Magdalena Kucia ◽  
Janina Ratajczak ◽  
...  
Keyword(s):  
Stem Cell ◽  
Purinergic Signaling ◽  
Negative Regulator ◽  
Extracellular Nucleotides ◽  
Heme Oxygenase 1 ◽  
Dependent Manner ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Pannexin 1 ◽  
Poor Mobilizers

Abstract Background . One of the problems with the mobilization of hematopoietic stem/progenitor cells (HSPCs) in the clinic is that a significant number of patients are poor mobilizers. In order to develop more efficient mobilization strategies, we have to better understand the mobilization process at the molecular and cellular levels. We have reported that HSPCs express heme oxygenase 1 (HO-1), which is a negative regulator of the complement cascade (ComC), which in turn is required for stem cell mobilization (Stem Cell Rev. 2015, 11:110-8, Leukemia. 2017, 31:446-458). Moreover, very recently we became interested in the novel role of extracellular nucleotides (EXNs) and nucleosides in this process (Leukemia 2018, in press, doi: 10.1038/s41375-018-0122-0). The most important EXN is adenosine triphosphate (ATP), which is involved in intracellular energy transfer but if released from activated cells into the extracellular space becomes an important signaling molecule involved in purinergic signaling. Secretion of ATP from activated cells occurs in a pannexin channel-dependent manner and is additionally augmented by a positive regulatory loop after ATP binding to the P2X7 purinergic receptor. We recently found that the ATP concentration during the mobilization process increases to micromolar concentrations in the BM microenvironment. Moreover, we found that in the extracellular BM space ATP is processed by the CD39 and CD73 ectonucleotidases to ADP, AMP, and finally to adenosine, which is also an important mediator of purinergic signaling. Hypothesis. We hypothesized that EXNs and purinergic signaling via extracellular ATP and its metabolite adenosine are involved in triggering the mobilization of HSPCs and that this effect is mediated by the expression of HO-1 in HSPCs. Materials and Methods. To address this question, 2-month-old P2X7-/-, CD39-/-, and CD73-/-mice as well as their normal wild type (WT) littermates were mobilized with G-CSF or AMD3100. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. The secretion of ATP from BM cells was inhibited by employing the pannexin 1 blocking drug probenecid or a synthetic pannexin 1 blocking peptide. In parallel, we evaluated i) the expression of HO-1 in HSPCs by employing RQ-PCR and western blot analysis and ii) the activation of the ComC by C5a ELISA. Results. Here we provide evidence that ATP, as an EXN secreted in a pannexin-1-dependent manner from BM cells, triggers activation of the ComC and initiates mobilization of HSPCs. Inhibition of the pannexin 1 channel by probenecid or a pannexin 1 blocking peptide inhibited this process. Furthermore, mobilization of HSPCs was augmented in a P2X7 receptor-dependent manner, evidenced by the finding that P2X7-/- mice were poor mobilizers. Furthermore, ATP is processed to adenosine in the extracellular space by CD39 and CD73 ectonucleotidases, and to our surprise we observed that CD73-deficient mice mobilize more HSPCs than their control wild type (WT) littermates, which indicates a novel negative role for adenosine in the mobilization process. This finding was confirmed by injecting mice with adenosine during AMD3100 administration. Finally, we found that ATP and adenosine modulate the mobilization process by regulating in opposite ways HSPC expression of HO-1, which is a negative regulator of mobilization. Therefore, while ATP downregulates expression of HO-1 in HSPCs and enhances mobilization, adenosine upregulates its expression and negatively affects the egress of HSPCs from BM into PB. Conclusions. We demonstrate for the first time that purinergic signaling involving ATP and its metabolite adenosine regulate the mobilization of HSPCs in an HO-1-dependent manner. While ATP triggers and promotes this process, adenosine has an inhibitory effect. The opposite effects of ATP and adenosine on the mobilization process can be explained by their different effects on the regulation of HO-1 expression in HSPCs. Finally, administration of ATP as a signaling molecule (together with G-CSF or AMD3100), inhibition of CD73 or HO-1 by small-molecule antagonists may provide the basis for more efficient mobilization strategies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Extracellular Adenosine Triphosphate (eATP) and Its Metabolite, Extracellular Adenosine (eAdo), as Opposing “Yin–Yang” Regulators of Nlrp3 Inflammasome in the Trafficking of Hematopoietic Stem/Progenitor Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Mariusz Z. Ratajczak ◽  
Magda Kucia
Keyword(s):  
Progenitor Cells ◽  
Adenosine Triphosphate ◽  
Nlrp3 Inflammasome ◽  
Protein Complex ◽  
Purinergic Signaling ◽  
Hematopoietic Cells ◽  
Sterile Inflammation ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Extracellular Adenosine

Nlrp3 inflammasome plays a pleiotropic role in hematopoietic cells. On the one hand, physiological activation of this intracellular protein complex is crucial to maintaining normal hematopoiesis and the trafficking of hematopoietic stem progenitor cells (HSPCs). On the other hand, its hyperactivation may lead to cell death by pyroptosis, and prolonged activity is associated with sterile inflammation of the BM and, as a consequence, with the HSPCs aging and origination of myelodysplasia and leukemia. Thus, we need to understand better this protein complex’s actions to define the boundaries of its safety window and study the transition from being beneficial to being detrimental. As demonstrated, the Nlrp3 inflammasome is expressed and active both in HSPCs and in the non-hematopoietic cells that are constituents of the bone marrow (BM) microenvironment. Importantly, the Nlrp3 inflammasome responds to mediators of purinergic signaling, and while extracellular adenosine triphosphate (eATP) activates this protein complex, its metabolite extracellular adenosine (eAdo) has the opposite effect. In this review, we will discuss and focus on the physiological consequences of the balance between eATP and eAdo in regulating the trafficking of HSPCs in an Nlrp3 inflammasome-dependent manner, as seen during pharmacological mobilization from BM into peripheral blood (PB) and in the reverse mechanism of homing from PB to BM and engraftment. We propose that both mediators of purinergic signaling and the Nlrp3 inflammasome itself may become important therapeutic targets in optimizing the trafficking of HSPCs in clinical settings.

scholarly journals Pannexin-1 channel “fuels” by releasing ATP from bone marrow cells a state of sterile inflammation required for optimal mobilization and homing of hematopoietic stem cells

2020 ◽  
Vol 16 (3) ◽  
pp. 313-325
Author(s):  
Monika Cymer ◽  
Katarzyna Brzezniakiewicz-Janus ◽  
Kamila Bujko ◽  
Arjun Thapa ◽  
Janina Ratajczak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Sterile Inflammation ◽  
Hematopoietic Stem ◽  
Extracellular Adenosine ◽  
Pannexin 1 ◽  
Molecular Pattern ◽  
Channel Blockage

Abstract An efficient harvest of hematopoietic stem/progenitor cells (HSPCs) after pharmacological mobilization from the bone marrow (BM) into peripheral blood (PB) and subsequent proper homing and engraftment of these cells are crucial for clinical outcomes from hematopoietic transplants. Since extracellular adenosine triphosphate (eATP) plays an important role in both processes as an activator of sterile inflammation in the bone marrow microenvironment, we focused on the role of Pannexin-1 channel in the secretion of ATP to trigger both egress of HSPCs out of BM into PB as well as in reverse process that is their homing to BM niches after transplantation into myeloablated recipient. We employed a specific blocking peptide against Pannexin-1 channel and noticed decreased mobilization efficiency of HSPCs as well as other types of BM-residing stem cells including mesenchymal stroma cells (MSCs), endothelial progenitors (EPCs), and very small embryonic-like stem cells (VSELs). To explain better a role of Pannexin-1, we report that eATP activated Nlrp3 inflammasome in Gr-1+ and CD11b+ cells enriched for granulocytes and monocytes. This led to release of danger-associated molecular pattern molecules (DAMPs) and mitochondrial DNA (miDNA) that activate complement cascade (ComC) required for optimal egress of HSPCs from BM. On the other hand, Pannexin-1 channel blockage in transplant recipient mice leads to a defect in homing and engraftment of HSPCs. Based on this, Pannexin-1 channel as a source of eATP plays an important role in HSPCs trafficking.

A Novel Underappreciated Role for the Extracellular Adenosine Triphosphate (ATP)-P2X4 Purinergic Receptor Axis in the Homing and Engraftment of HSPCs

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
Mateusz Adamiak ◽  
Arjun Thapa ◽  
Kamila Bujko ◽  
Valentina Pensato ◽  
Magdalena Kucia ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Adenosine Triphosphate ◽  
Nlrp3 Inflammasome ◽  
Purinergic Signaling ◽  
Blood Platelets ◽  
The Body ◽  
Recipient Mouse ◽  
P2x7 Receptors ◽  
Hematopoietic Stem

Background. Adenosine triphosphate (ATP) is an important nucleotide involved in intracellular energy transfer, but when released from activated cells into the extracellular space as extracellular ATP (eATP) it becomes a crucial mediator of the purinergic signaling network. Purinergic receptors for extracellular nucleotides (EXNs), expressed on the surface of all cells in the body, are represented by the P1, P2X, and P2Y receptor families, which are among the most abundant receptors in living organisms. Of all these receptors, the P2X receptor family is most highly specific for eATP signaling and consists of seven members (P2X1-7). We found that human and murine hematopoietic stem progenitor cells (HSPCs) highly express two members of this family, the P2X4 and P2X7 receptors. We recently reported that both are involved in optimal mobilization of HSPCs by activating Nlrp3 inflammasome (Leukemia 2020 Jun;34(6):1512-1523 and Stem Cell Rev Rep. 2019 Jun;15(3):391-403). We also reported that the P2X7 receptor expressed on the surface of HSPCs facilitates the homing and engraftment of HSPCs by increasing their responsiveness to SDF-1 gradients. Interestingly, it has been proposed that both receptors heterodimerize to exert optimal activity. Hypothesis. Since, the P2X4 and P2X7 receptors show several similar biological effects in non-hematopoietic cells, we became interested in the role of the P2X4 receptor in homing and engraftment of HSPCs.Materials and Methods. To test this hypothesis, we isolated SKL cells from P2X4-KO mice and tested them for migration in response to BM chemoattractants, including the major homing factor SDF-1. Next, we tested the short- and long-term homing of mouse BM cells after exposure to the P2X4-specific inhibitor PBS12054 in normal mice by evaluating the number of donor-derived PKH67-labeled BMMNCs and CFU-GM clonogenic progenitors isolated from recipient mouse BM 24 hours after transplantation as well as the number of day-12 colony-forming units in spleen (CFU-S) and day-12 CFU-GM clonogenic progenitors. These data were confirmed in transplant studies employing P2X4-KO bone marrow cells. In parallel, we also evaluated the recovery kinetics of leukocytes and blood platelets in the PB of transplanted animals. Finally, we also perturbed P2X4 expression in transplanted mice with PBS12054 and studied the effect on homing and engraftment of normal BM cells, as described above. Results. We found that P2X4-KO mouse HSPCs have a defect in migration in response to BM chemoattractants involved in BM homing, including the major homing factor SDF-1 as well as the supportive factors S1P and eATP. Perturbation of P2X4 expression on the surface of HSPCs led to significant defective homing and engraftment of HSPCs. Moreover, inhibition of P2X4 in the recipient mouse BM microenvironment had a similar effect. Conclusions. We identified for the first time the role of eATP-P2X4 signaling in the homing and engraftment of HSPCs. To explain this result, we conclude that the eATP-P2X4 axis is, like the eATP-P2X7 axis, a potent activator of Nlrp3 inflammasomes and that defective eATP-P2X4 signaling impairs the role of purinergic signaling and the Nlrp3 inflammasome in homing and engraftment. Moreover, our results show a similar homing and engraftment phenotype for P2X4-KO mice as that seen in P2X7-KO animals, which provides functional support for the proposed dimerization of P2X7 with P2X4 receptors and the necessary presence of both receptors for optimal function. This question is currently being addressed in our laboratory by employing the fluorescence resonance energy transfer (FRET) technique. Finally, we provide additional evidence that, in addition to SDF-1 and S1P, eATP and purinergic signaling involving P2X4 and P2X7 receptors is an important and underappreciated regulator of HSPC trafficking and a potential target for molecular optimization of both processes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Administration of Plerixafor in Poor Mobilizers Allows Mobilization of Haematopoietic Progenitors: Clonogenic Potential and Engraftment Analysis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5633-5633
Author(s):  
Laura Pezzetti ◽  
Giambattista Bertani ◽  
Liliana Intropido ◽  
Tiziana Lamacchia ◽  
Mariateresa Pugliano ◽  
...  
Keyword(s):  
Stem Cell ◽  
Conflicts Of Interest ◽  
Biological Data ◽  
Hematopoietic Stem ◽  
Group A ◽  
Stromal Cell Derived Factor ◽  
Semi Solid ◽  
N 93 ◽  
Group B ◽  
Poor Mobilizers

BACKGROUND Plerixafor (PLX) blocks the binding of stromal cell-derived factor to CXCR4, resulting in hematopoietic stem cell (HSC) release from the bone marrow. It has been successfully used as a mobilizing agent in poor mobilizers (PM). The best strategy is probably the "pre-emptive" (on demand) use, as it allows an "on time" identification of PMs, preventing collection failure and need for further mobilization. Usually a cut-off of < 10/ μL CD34+ cell count at leukocyte recovery is used for PLXa dministration, while a minimum level is not defined, as mobilization may be obtained even with very low values. An important issue that has not been extensively explored so far is represented by the biological characteristics of the yields collected with PLX, especially clonogenicity, and its clinical counterpart, represented by the engraftment times observed after transplant. In this report we retrospectively analysed our data on the use of PLX in PMs, evaluated its efficacy and focused on the clonogenicity of collected stem cells and engraftment post auto transplant (ASCT). METHODS We collected data on PM patients mobilized with PLX between 2011 and 2019:Clinical data: mobilization and collection performance, transplant rate and engraftment times (days to PMN > 500/uL).Biological data: Clonogenicity of HSC collected after PLX, measured by hematopoietic progenitor semi-solid cultures according to Stem Cell Technology. These clinical and biological data were then compared to those obtained in patients mobilized without PLX and in allogeneic donors who received G-CSF in the same period. RESULTS From January 2011 to June 2019 PLX was used in 73 mobilization cycles, performed in 64 patients. Patients characteristics are described in Table 1. 105 doses of PLX were given during 73 mobilization cycles (1,44 doses/cycle) and 93 collections performed (1,27/ cycle). Circulating CD34+ pre PLX administration were in median 6,81/µl (range: 0,27-21), while after treatment 26,20/µl (range: 4,8-155,4). Using a cut-off of ≥ 10/ μL CD34+, a successful mobilization was achieved in 67/73 cycles (success rate 91,7%). Overall 59 out of 64 patients achieved the collection target (92,2%) at any time and 54/64 have been transplanted (84,4%), the other 10 not yet due to mobilization failure (n=2), insufficient yield (n=3), clinical unfitness (n=2) or because too early (n=3). A median of 4 HPC bags were reinfused (range: 2-12) and median time to WBC engraftment was 10 days (8-21). Engrafment times are in line with those of MM and NHL patients mobilized without PLX in the same period of time (median: 10, range: 8-12) at our Institution. Interestingly, 33 out of 35 pts mobilized with PLX (94,2%), compared to 442/449 (98,4%) engrafted in ≤11 days. We analyzed the cellular composition of yields and clonogenicity of HSC collected in 3 different groups:Group A: PLX mobilization (n=93)Group B: chemo + G-CSF without PLX (n=755)Group C: G-CSF mobilization in allogeneic donors (n=206) We failed to demonstrate a statistical difference between clonogenicity in group A and B (p=0,691), while clonogenicity was slightly but significantly higher in group C compared to group A and B (p< 0,001). Data are shown in tables 2,3 and 4. CONCLUSION In our cohort of patients, administration of PLX in PM resulted in successful mobilization of HPCs with good clonogenicity and engraftment potential. The use of PLX allowed an high proportion of patients to undergo ASCT. Disclosures No relevant conflicts of interest to declare.

scholarly journals Novel Evidence That Alternative Pathway of Complement Cascade Activation is Required for Optimal Homing and Engraftment of Hematopoietic Stem/progenitor Cells

2022 ◽  
Author(s):  
Mateusz Adamiak ◽  
Andrzej Ciechanowicz ◽  
Vira Chumak ◽  
Kamila Bujko ◽  
Janina Ratajczak ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Purinergic Signaling ◽  
Alternative Pathway ◽  
Sterile Inflammation ◽  
Complement Cascade ◽  
Hematopoietic Stem ◽  
Alternative Pathway Of Complement ◽  
Endothelial Migration ◽  
Lethal Irradiation

AbstractWe reported in the past that activation of the third (C3) and fifth element (C5) of complement cascade (ComC) is required for a proper homing and engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs). Since myeloablative conditioning for transplantation triggers in recipient bone marrow (BM) state of sterile inflammation, we have become interested in the role of complement in this process and the potential involvement of alternative pathway of ComC activation. We noticed that factor B deficient mice (FB-KO) that do not activate properly alternative pathway, engraft poorly with BM cells from normal wild type (WT) mice. We observed defects both in homing and engraftment of transplanted HSPCs. To shed more light on these phenomena, we found that myeloablative lethal irradiation conditioning for transplantation activates purinergic signaling, ComC, and Nlrp3 inflammasome in WT mice, which is significantly impaired in FB-KO animals. Our proteomics analysis revealed that conditioned for transplantation lethally irradiated FB-KO compared to normal control animals have lower expression of several proteins involved in positive regulation of cell migration, trans-endothelial migration, immune system, cellular signaling protein, and metabolic pathways. Overall, our recent study further supports the role of innate immunity in homing and engraftment of HSPCs. Graphical Abstract

scholarly journals Knockout of P2rx7 purinergic receptor attenuates cyst growth in a rat model of ARPKD

2019 ◽  
Vol 317 (6) ◽  
pp. F1649-F1655 ◽  
Author(s):  
Sergey N. Arkhipov ◽  
D’Anna L. Potter ◽  
Aron M. Geurts ◽  
Tengis S. Pavlov
Keyword(s):  
Purinergic Receptor ◽  
Kidney Diseases ◽  
Purinergic Signaling ◽  
Experimental Studies ◽  
Sodium Reabsorption ◽  
Channel Activity ◽  
P2x7 Receptors ◽  
Exon 2 ◽  
Pannexin 1 ◽  
Cyst Growth

The severity of polycystic kidney diseases (PKD) depends on the counterbalancing of genetic predisposition and environmental factors exerting permissive or protective influence on cyst development. One poorly characterized phenomenon in the cystic epithelium is abnormal purinergic signaling. Earlier experimental studies revealed the high importance of the ionotropic P2X receptors (particularly, P2X7) in the pathophysiology of the cyst wall. To study mechanisms of P2X7 involvement in cyst growth and aspects of targeting these receptors in PKD treatment we performed a CRISPR/SpCas9-mediated global knockout of the P2rx7 gene in PCK rats, a model of autosomal recessive PKD (ARPKD). A single base insertion in exon 2 of the P2rx7 gene in the renal tissues of homozygous mutant animals leads to lack of P2X7 protein that did not affect their viability or renal excretory function. However, PCK. P2rx7 rats demonstrated slower cyst growth (but not formation of new cysts) compared with heterozygous and PCK. P2rx7+ littermates. P2X7 receptors are known to activate pannexin-1, a plasma channel capable of releasing ATP, and we found here that pannexin-1 expression in the cystic epithelium is significantly higher than in nondilated tubules. P2X7 deficiency reduces renal pannexin-1 protein expression and daily urinary ATP excretion. Patch-clamp analysis revealed that lack of P2X7 increases epithelial sodium channel activity in renal tissues and restores impaired channel activity in cysts. Interpretation of our current data in the context of earlier studies strongly suggests that P2X7 contributes to cyst growth by increasing pannexin-1-dependent pathogenic ATP release into the lumen and reduction of sodium reabsorption across the cyst walls.

scholarly journals Novel Evidence That Extracellular Adenosine Triphosphate (ATP), As a Purinergic Signaling Mediator, Activates Mobilization By Engaging a P2X4 Ligand-Gated Cation Channel Receptor Expressed on the Surface of Hematopoietic and Innate Immunity Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4472-4472 ◽  
Author(s):  
Kamila Bujko ◽  
Mateusz Adamiak ◽  
Arjun Thapa ◽  
Magdalena Kucia ◽  
Janina Ratajczak ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Adenosine Triphosphate ◽  
Small Molecule ◽  
Nlrp3 Inflammasome ◽  
P2x7 Receptor ◽  
Purinergic Signaling ◽  
Hematopoietic Cells ◽  
Wild Type ◽  
P2x4 Receptor ◽  
Molecule Inhibitor

Background . Adenosine triphosphate (ATP) is an important nucleotide involved in intracellular energy transfer, but it is also released from activated cells into the extracellular space as a crucial component of the purinergic signaling network. Purinergic signaling is an ancient form of extracellular signaling that is mediated by ATP and other extracellular nucleotides (EXNs). Purinergic receptors for EXNs are expressed on the surface of all cells in the body; are represented by several families of P1, P2X, and P2Y receptors; and are among the most abundant receptors in living organisms. Of all these receptors, the P2X receptor family is most highly specific for ATP signaling and consists of seven members (P2X1-7). We recently reported that by activating the P2X7 receptor ATP activates the Nlrp3 inflammasome in hematopoietic cells and significantly enhances G-CSF-induced mobilization of hematopoietic stem progenitor cells (HSPCs, Leukemia 2018, 32:1920-1931). Hypothesis. Since P2X7-KO mice still mobilize some HSPCs, we hypothesized that, in addition to the P2X7 receptor, other receptors from this family also play a role in the mobilization process.Materials and Methods. RT-PCR and FACS were employed to phenotype human and murine bone marrow (BM) mononuclear cells (MNCs) as well as sorted HSCs for expression of P2X receptors. Mobilization studies were performed in wild type mice in which the P2X4 receptor was blocked by the specific small-molecule inhibitor PSB12054 and in P2X7-KO animals. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. We also evaluated activation of the Nlrp3 inflammasome as well as other inflammasomes expressed in hematopoietic cells, including Aim2, Nlrp1a, Nlrp1b, and Nlrc4. Results. We found that, of all the receptors of the P2X family, two members, P2X7 and P2X4, are highly expressed on the surface of murine and human MNCs and HSPCs (Figure 1a). Activation of both receptors on the surface of innate immunity CD11b+Gr-1+ cells resulted in activation of the Nlrp3, Nlrp1a, Nlrp1b, and Aim2 inflammasomes. Inhibition of the P2X4 receptor by a specific small-molecule inhibitor decreased G-CSF- and AMD3100-induced mobilization in wild type mice (Figure 1b). Interestingly, we observed differences during AMD3100 mobilization, namely that while P2X7-KO deficiency resulted in decreased G-CSF-induced mobilization and did not affect AMD3100 mobilization efficiency, P2X4 inhibition, by contrast, led to a profound decrease in both G-CSF- and AMD3100-induced mobilization efficiency. Conclusions. Our results further support an important role for purinergic signaling in the mobilization of HSPCs. We also demonstrated that P2X7 and P2X4 receptors, which are highly expressed on human and murine hematopoietic cells, are involved in G-CSF-induced mobilization by activating the Nlrp3, Nlrp1a, Nlrp1b and Aim2 inflammasomes. By contrast, AMD3100-induced mobilization requires expression of P2X4 but not P2X7 receptors, which implies the involvement of different mechanisms and indicates that P2X7 deficiency can be compensated by the P2X4 receptor. Currently, we are performing RNASeq studies to identify differences in the G-CSF- and AMD3100-induced pathways in the mobilization process. These investigations will shed more light on the molecular pathways involved in the egress of HSPCs from BM into PB and will help to design better mobilization protocols in cases of poor mobilizers. Figure 1 Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document