P2X7R and P2X4R expression of mice submandibular gland in high-fat diet/streptozotocin-induced type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is a chronic inflammatory disease that can compromise the functioning of various organs, including the salivary glands (SG). The purinergic system is one of the most important inflammatory pathways in T2DM condition, and P2X7R and P2X4R are the primary purinergic receptors in SG that regulate inflammatory homeostasis. This study aimed to evaluate P2X7R and P2X4R expression, and morphological changes in the submandibular gland (SMG) in T2DM. Twenty-four 5-week-old mice were randomly assigned to control (CON) and diabetes mellitus (DM) groups (n = 12 each). Body weight, diet, and blood glucose levels were monitored weekly. The histomorphology of the SMG and the expression of the P2X7R, and P2X7R was evaluated by immunohistochemistry (IHC) staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) at 11 and 13 weeks of age. Our findings indicate a significant increase in food consumption, body weight, and blood glucose levels in the DM group. Although a significant increase in P2X7R and P2X4R expression was observed in the DM groups, the receptor location remained unchanged. We also observed a significant increase in the acinar area in the DM13w group, and a significant decrease in the ductal area in the DM11w and DM13w groups. Targeting purinergic receptors may offer novel therapeutic methods for diabetic complications.

Deficiency of purinergic P2X4 receptor alleviates experimental autoimmune hepatitis in mice.

Purinergic P2X4 receptor (P2X4R) has been shown to have immunomodulatory properties in infection, inflammation, and organ damage including liver regeneration and fibrosis. However, the mechanisms and pathophysiology associated with P2X4R during acute liver injury remain unknown. We used P2X4R-/- mice to explore the role of P2X4R in three different models of acute liver injury caused by concanavalin A (ConA), carbon tetrachloride, and acetaminophen. ConA treatment results in an increased expression of P2X4R in the liver of mice, which was positively correlated with higher levels of aspartate aminotransferase and alanine aminotransferase in the serum. However, P2X4R gene ablation significantly reduced the severity of acute hepatitis in mice caused by ConA, but not by carbon tetrachloride or acetaminophen. The protective benefits against immune-mediated acute hepatitis were achieved via modulating inflammation (Interleukin (IL)-1ß, IL-6, IL-17A, interferon-γ, tumor necrosis factor-α), oxidative stress (malondialdehyde, superoxide dismutase, glutathione peroxidase, and catalase), apoptosis markers (Bax, Bcl-2, and Caspase-3), autophagy biomarkers (LC3, Beclin-1, and p62), and nucleotide oligomerization domain-likereceptorprotein 3(NLRP3) inflammasome-activated pyroptosis markers (NLRP3, Gasdermin D, Caspase-1, ASC, IL-1ß). Additionally, administration of P2X4R antagonist (5-BDBD) or agonist (cytidine 5'-triphosphate) either improved or worsened ConA-induced autoimmune hepatitis, respectively. This study is the first to reveal that the absence of the P2X4 receptor may mitigate immune-mediated liver damage, potentially by restraining inflammation, oxidation, and programmed cell death mechanisms. And highlight P2X4 receptor is essential for ConA-induced acute hepatitis.

Methods for studying P2X4 receptor ion channels in immune cells.

The P2X4 receptor is a trimeric ligand-gated ion channel activated by adenosine 5'-triphosphate (ATP). P2X4 is present in immune cells with emerging roles in inflammation and immunity, and related disorders. This review aims to provide an overview of the methods commonly used to study P2X4 in immune cells, focusing on those methods used to assess P2RX4 gene expression, the presence of the P2X4 protein, and P2X4 ion channel activity in these cells from humans, dogs, mice and rats. P2RX4 gene expression in immune cells is commonly assessed using semi-quantitative and quantitative reverse-transcriptase-PCR. The presence of P2X4 protein in immune cells is mainly assessed using anti-P2X4 polyclonal antibodies with immunoblotting or immunochemistry, but the use of these antibodies, as well as monoclonal antibodies and nanobodies to detect P2X4 with flow cytometry is increasing. Notably, use of an anti-P2X4 monoclonal antibody and flow cytometry has revealed that P2X4 is present on immune cells with a rank order of expression in eosinophils, then neutrophils and monocytes, then basophils and B cells, and finally T cells. P2X4 ion channel activity has been assessed mainly by Ca2+ flux assays using the cell permeable Ca2+-sensitive dyes Fura-2 and Fluo-4 with fluorescence microscopy, spectrophotometry, or flow cytometry. However, other methods including electrophysiology, and fluorescence assays measuring Na+ flux (using sodium green tetra-acetate) and dye uptake (using YO-PRO-12+) have been applied. Collectively, these methods have demonstrated the presence of functional P2X4 in monocytes and macrophages, microglia, eosinophils, mast cells and CD4+ T cells, with other evidence suggestive of functional P2X4 in dendritic cells, neutrophils, B cells and CD8+ T cells.

Naringin inhibits P2X4 receptor expression on satellite glial cells in the neonatal rat dorsal root ganglion.

Naringin inhibits inflammation and oxidative stress, the P2 purinoreceptor X4 receptor (P2X4R) is associated with glial cell activation and inflammation, the purpose of this study is to investigate the effects of naringin on P2X4 receptor expression on satellite glial cells (SGCs) and its possible mechanisms. ATP promoted the SGC activation and upregulated P2X4R expression; naringin inhibited SGC activation, decreased expression of P2X4R, P38 MAPK/ERK, and NF-κB, and reduced levels of Ca2+, TNF-α, and IL-1ß in SGCs in an ATP-containing environment. These findings suggest that naringin attenuates the ATP-induced SGC activation and reduces P2X4R expression via the Ca2+-P38 MAPK/ERK-NF-κB pathway.

Structural insights into the allosteric inhibition of P2X4 receptors.

P2X receptors are ATP-activated cation channels, and the P2X4 subtype plays important roles in the immune system and the central nervous system, particularly in neuropathic pain. Therefore, P2X4 receptors are of increasing interest as potential drug targets. Here, we report the cryo-EM structures of the zebrafish P2X4 receptor in complex with two P2X4 subtype-specific antagonists, BX430 and BAY-1797. Both antagonists bind to the same allosteric site located at the subunit interface at the top of the extracellular domain. Structure-based mutational analysis by electrophysiology identified the important residues for the allosteric inhibition of both zebrafish and human P2X4 receptors. Structural comparison revealed the ligand-dependent structural rearrangement of the binding pocket to stabilize the binding of allosteric modulators, which in turn would prevent the structural changes of the extracellular domain associated with channel activation. Furthermore, comparison with the previously reported P2X structures of other subtypes provided mechanistic insights into subtype-specific allosteric inhibition.

Purinergic neurotransmission receptor P2X4 silencing alleviates intracerebral hemorrhage-induced neuroinflammation by blocking the NLRP1/Caspase-1 pathway.

This study is performed to explore the role of P2X4 in intracerebral hemorrhage (ICH) and the association between P2X4 and the NLRP1/Caspase-1 pathway. The mouse ICH model was established via collagenase injection into the right basal ganglia. P2X4 expression in brain tissues was knocked down via intracerebroventricular injection with adeno-associated virus (AAV) harboring shRNA against shP2X4. The gene expression of P2X4 and protein levels related to NLRP1 inflammasome were detected using qRT-PCR and Western blot analysis, respectively. Muramyl dipeptide (an activator of NLRP1) was used to activate NLRP1 in brain tissues. ICH induced high expression of P2X4 in mouse brain tissues. The knockdown of P2X4 alleviated short- and long-term neurological deficits of ICH mice, as well as inhibited the tissue expression and serum levels of pro-inflammatory cytokines, including TNF-α, interleukin (IL)-6, and IL-1ß. Additionally, the expressions of NLRP1, ASC, and pro-Caspase-1 were down-regulated upon P2X4 silencing. Moreover, neurological impairment and the expression and secretion of cytokines after P2X4 silencing were aggravated by the additional administration of MDP. P2X4 knockdown represses neuroinflammation in brain tissues after ICH. Mechanistically, P2X4 inhibition exerts a neuroprotective effect in ICH by blocking the NLRP1/Caspase-1 pathway.

Transcriptomic analysis reveals novel age-independent immunomodulatory proteins as a mode of cerebroprotection in P2X4 receptor knockout mice after ischemic stroke.

Identification of new potential drug target proteins and their plausible mechanisms for stroke treatment is critically needed. We previously showed that genetic deletion and short-term pharmacological inhibition of P2X4, a purinergic receptor for adenosine triphosphate (ATP), provides acute cerebroprotection. However, potential mechanisms remain unknown. Therefore, we employed RNA-Seq technology to identify the gene expression profiles and pathway analysis followed by qPCR validation of differentially expressed genes (DEGs). This analysis identified roles of DEGs in certain biological processes responsible for P2X4R-dependent cerebroprotection after stroke. We subjected both young and aged male and female global P2X4 receptor knock out (P2X4RKO) and littermate WT (WT) mice to ischemic stroke. After three days, mice were sacrificed, and total RNA was isolated using Trizol and subjected to RNA-Seq and NanoString-mediated qPCR. DESeq2, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) were used to identify gene expression profiles and biological pathways. We found 2246 DEGs in P2X4R KO vs. WT tissue after stroke. Out of these DEGs, 1920 genes were downregulated and 325 genes were upregulated in P2X4R KO. GO/IPA analysis of the top 300 DEGs suggests an enrichment of inflammation and extracellular matrix component genes. qPCR validation of the top 30 DEGs revealed downregulation of two common age-independent genes in P2X4R KO mice: Interleukin-6 (Il-6), an inflammatory cytokine, and Cytotoxic T Lymphocyte-Associated Protein 2 alpha (Ctla2a), an immunosuppressive factor. These data suggest that P2X4R-mediated cerebroprotection after stroke is initiated by attenuation of immune modulatory pathways in both young and aged mice of both sexes.

Imperatorin Improves Obesity-Induced Cardiac Sympathetic Nerve Injury Mediated by P2X4 Receptor in Stellate Sympathetic Ganglion.

Obesity can activate the inflammatory signal pathway, induce in the body a state of chronic inflammation, and increase the excitability of the sympathetic nervous system, which may induce sympathetic neuropathic injury. The stellate sympathetic ganglia (SG) can express the P2X4 receptor, and the abnormal expression of the P2X4 receptor is related to inflammation. Imperatorin (IMP) is a kind of furan coumarin plant which has anti-inflammatory effects. This project aimed to investigate whether IMP can affect the expression of P2X4 receptors in the SG of obese rats to display a protective effect from high-fat-triggered cardiac sympathetic neuropathic injury. Molecular docking through homology modelling revealed that IMP had good affinity for the P2X4 receptor. Our results showed that compared with the normal group, the administration of IMP or P2X4 shRNA decreased sympathetic excitement; reduced the serum levels of triglyceride, total cholesterol, and lactate dehydrogenase; downregulated the expression of P2X4 receptors in SG; and inhibited the expression of inflammatory factors in the SG and serum of obese rats significantly. In addition, the expression of factors associated with the cell pyroptosis GSDMD, caspase-1, NLRP-3, and IL-18 in obese rats were significantly higher than those of the normal rats, and such effects were decreased after treatment with IMP or P2X4 shRNA. Furthermore, IMP significantly reduced the ATP-activated currents in HEK293 cells transfected with P2X4 receptor. Thus, the P2X4 receptor may be a key target for the treatment of obesity-induced cardiac sympathetic excitement. IMP can improve obesity-induced cardiac sympathetic excitement, and its mechanism of action may be related to the inhibition of P2X4 receptor expression and activity in the SG, suppression of cellular pyroptosis in the SG, and reduction of inflammatory factor levels.

Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy.

The purinergic receptor P2X7 (P2X7R) is implicated in all neurodegenerative diseases of the central nervous system. It is also involved in the retinal degeneration associated with glaucoma, age-related macular degeneration, and diabetic retinopathy, and its overexpression in the retina is evident in these disorders. Retinitis pigmentosa is a progressive degenerative disease that ultimately leads to blindness. Here, we investigated the expression of P2X7R during disease progression in the rd10 mouse model of RP. As the purinergic receptor P2X4 is widely co-expressed with P2X7R, we also studied its expression in the retina of rd10 mice. The expression of P2X7R and P2X4R was examined by immunohistochemistry, flow cytometry, and western blotting. In addition, we analyzed retinal functionality by electroretinographic recordings of visual responses and optomotor tests and retinal morphology. We found that the expression of P2X7R and P2X4R increased in rd10 mice concomitant with disease progression, but with different cellular localization. Our findings suggest that P2X7R and P2X4R might play an important role in RP progression, which should be further analyzed for the pharmacological treatment of inherited retinal dystrophies.

Increased surface P2X4 receptors by mutant SOD1 proteins contribute to ALS pathogenesis in SOD1-G93A mice.

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron (MN) disease characterized by protein misfolding and aggregation leading to cellular degeneration. So far neither biomarker, nor effective treatment has been found. ATP signaling and P2X4 receptors (P2X4) are upregulated in various neurodegenerative diseases. Here we show that several ALS-related misfolded proteins including mutants of SOD1 or TDP-43 lead to a significant increase in surface P2X4 receptor density and function in vitro. In addition, we demonstrate in the spinal the cord of SOD1-G93A (SOD1) mice that misfolded SOD1-G93A proteins directly interact with endocytic adaptor protein-2 (AP2); thus, acting as negative competitors for the interaction between AP2 and P2X4, impairing constitutive P2X4 endocytosis. The higher P2X4 surface density was particularly observed in peripheral macrophages of SOD1 mice before the onset and during the progression of ALS symptoms positioning P2X4 as a potential early biomarker for ALS. P2X4 expression was also upregulated in spinal microglia of SOD1 mice during ALS and affect microglial inflammatory responses. Importantly, we report using double transgenic SOD1 mice expressing internalization-defective P2X4mCherryIN knock-in gene or invalidated for the P2X4 gene that P2X4 is instrumental for motor symptoms, ALS progression and survival. This study highlights the role of P2X4 in the pathophysiology of ALS and thus its potential for the development of biomarkers and treatments. We also decipher the molecular mechanism by which misfolded proteins related to ALS impact P2X4 trafficking at early pathological stage in cells expressing-P2X4.

Blocking P2X purinoceptor 4 signalling alleviates cigarette smoke induced pulmonary inflammation.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with elevated ATP levels in the extracellular space. Once released, ATP serves as danger signal modulating immune responses by activating purinergic receptors. Accordingly, purinergic signalling has been implicated in respiratory inflammation associated with cigarette smoke exposure. However, the role of P2X4-signalling has not been fully elucidated yet. METHODS: Here, we analysed the P2X4 mRNA expression in COPD patients as well as cigarette smoke-exposed mice. Furthermore, P2X4-signalling was blocked by either using a specific antagonist or genetic depletion of P2rx4 in mice applied to an acute and prolonged model of cigarette smoke exposure. Finally, we inhibited P2X4-signalling in macrophages derived from THP-1 before stimulation with cigarette smoke extract. RESULTS: COPD patients exhibited an increased P2X4 mRNA expression in cells isolated from the bronchoalveolar lavage fluid and peripheral mononuclear cells. Similarly, P2rx4 expression was elevated in lung tissue of mice exposed to cigarette smoke. Blocking P2X4-signalling in mice alleviated cigarette smoke induced airway inflammation as well as lung parenchyma destruction. Additionally, human macrophages derived from THP-1 cells released reduced concentrations of proinflammatory cytokines in response to cigarette smoke extract stimulation when P2X4 was inhibited. CONCLUSION: Taken together, we provide evidence that P2X4-signalling promotes innate immunity in the immunopathologic responses induced by cigarette smoke exposure.

MiR-106b-5p Attenuates Neuropathic Pain by Regulating the P2X4 Receptor in the Spinal Cord in Mice.

The P2X4 receptor (P2X4R) can be upregulated after nerve injury, and its mediated spinal microglial activation makes a critical contribution to pathologically enhanced pain processing in the dorsal horn. Although some studies have partly clarified the mechanism underlying altered P2X4R expression, the specific mechanism is not well understood. MicroRNAs (miRNAs) are small noncoding RNAs which control gene expression by binding with their target mRNAs. Thus, in the present study, we investigated whether miRNA is involved in the pathogenesis of neuropathic pain by regulating P2X4R. Our results showed that P2X4R was upregulated in the spinal dorsal horn of mice following spared nerve injury (SNI), and 69 miRNAs (46 upregulated and 23 downregulated miRNAs) were differentially expressed (fold change > 2.0, P < 0.05). P2X4R was found to be a major target of miR-106b-5p (one of the downregulated miRNAs) using bioinformatics technology; quantitative real-time PCR analysis confirmed the change in expression of miR-106b-5p, and dual-luciferase reporter assays confirmed the correlation between them. Fluorescence in situ hybridization was used to show cell co-localization of P2X4R and miR-106b-5p in the spinal dorsal horn. Transfection with miR-106b-5p mimic into BV2 cells reversed the upregulation of P2X4R induced by lipopolysaccharide (LPS). Moreover, miR-106b-5p overexpression significantly attenuated neuropathic pain induced by SNI, with decreased expression of P2X4R mRNA and protein in the spinal dorsal horn; intrathecal miR-106b-5p antagomir induced pain behaviors, and increased expression of P2X4R in the spinal dorsal horn of naïve mice. These data suggest that miR-106b-5p can serve as an important regulator of neuropathic pain development by targeting P2X4R.

The P2X4 Receptor: Cellular and Molecular Characteristics of a Promising Neuroinflammatory Target.

The adenosine 5'-triphosphate-gated P2X4 receptor channel is a promising target in neuroinflammatory disorders, but the ability to effectively target these receptors in models of neuroinflammation has presented a constant challenge. As such, the exact role of P2X4 receptors and their cell signalling mechanisms in human physiology and pathophysiology still requires further elucidation. To this end, research into the molecular mechanisms of P2X4 receptor activation, modulation, and inhibition has continued to gain momentum in an attempt to further describe the role of P2X4 receptors in neuroinflammation and other disease settings. Here we provide an overview of the current understanding of the P2X4 receptor, including its expression and function in cells involved in neuroinflammatory signalling. We discuss the pharmacology of P2X4 receptors and provide an overview of P2X4-targeting molecules, including agonists, positive allosteric modulators, and antagonists. Finally, we discuss the use of P2X4 receptor modulators and antagonists in models of neuroinflammatory cell signalling and disease.

P2x4 receptor promotes mammary cancer progression by sustaining autophagy and associated mesenchymal transition.

Metastatic progression is a major burden for breast cancer patients and is associated with the ability of cancer cells to overcome stressful conditions, such as nutrients deprivation and hypoxia, and to gain invasive properties. Autophagy and epithelial-to-mesenchymal transition are critical contributors to these processes. Here, we show that the P2X4 purinergic receptor is upregulated in breast cancer biopsies from patients and it is primarily localised in endolysosomes. We demonstrate that P2X4 enhanced invasion in vitro, as well as mammary tumour growth and metastasis in vivo. The pro-malignant role of P2X4 was mediated by the regulation of lysosome acidity, the promotion of autophagy and cell survival. Furthermore, the autophagic activity was associated with epithelial-to-mesenchymal transition (EMT), and this role of P2X4 was even more pronounced under metabolic challenges. Pharmacological and gene silencing of P2X4 inhibited both autophagy and EMT, whereas its rescue in knocked-down cells led to the restoration of the aggressive phenotype. Together, our results demonstrate a previously unappreciated role for P2X4 in regulating lysosomal functions and fate, promoting breast cancer progression and aggressiveness.

P2X4 deficiency reduces atherosclerosis and plaque inflammation in mice.

Extracellular adenosine-5'-triphosphate (ATP) acts as an import signaling molecule mediating inflammation via purinergic P2 receptors. ATP binds to the purinergic receptor P2X4 and promotes inflammation via increased expression of pro-inflammatory cytokines. Because of the central role of inflammation, we assumed a functional contribution of the ATP-P2X4-axis in atherosclerosis. Expression of P2X4 was increased in atherosclerotic aortic arches from low-density lipoprotein receptor-deficient mice being fed a high cholesterol diet as assessed by real-time polymerase chain reaction and immunohistochemistry. To investigate the functional role of P2X4 in atherosclerosis, P2X4-deficient mice were crossed with low-density lipoprotein receptor-deficient mice and fed high cholesterol diet. After 16 weeks, P2X4-deficient mice developed smaller atherosclerotic lesions compared to P2X4-competent mice. Furthermore, intravital microscopy showed reduced ATP-induced leukocyte rolling at the vessel wall in P2X4-deficient mice. Mechanistically, we found a reduced RNA expression of CC chemokine ligand 2 (CCL-2), C-X-C motif chemokine-1 (CXCL-1), C-X-C motif chemokine-2 (CXCL-2), Interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) as well as a decreased nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)-inflammasome priming in atherosclerotic plaques from P2X4-deficient mice. Moreover, bone marrow derived macrophages isolated from P2X4-deficient mice revealed a reduced ATP-mediated release of CCL-2, CC chemokine ligand 5 (CCL-5), Interleukin-1ß (IL-1ß) and IL-6. Additionally, P2X4-deficient mice shared a lower proportion of pro-inflammatory Ly6Chigh monocytes and a higher proportion of anti-inflammatory Ly6Clow monocytes, and expressend less endothelial VCAM-1. Finally, increased P2X4 expression in human atherosclerotic lesions from carotid endarterectomy was found, indicating the importance of potential implementations of this study's findings for human atherosclerosis. Collectively, P2X4 deficiency reduced experimental atherosclerosis, plaque inflammation and inflammasome priming, pointing to P2X4 as a potential therapeutic target in the fight against atherosclerosis.

The Purinergic Receptor P2X4 Promotes Th17 Activation and the Development of Arthritis.

Purinergic signaling plays a major role in T cell activation leading to IL-2 production and proliferation. However, it is unclear whether purinergic signaling contributes to the differentiation and activation of effector T cells. In this study, we found that the purinergic receptor P2X4 was associated with human Th17 cells but not with Th1 cells. Inhibition of P2X4 receptor with the specific antagonist 5-BDBD and small interfering RNA inhibited the development of Th17 cells and the production of IL-17 by effector Th17 cells stimulated via the CD3/CD28 pathway. Our results showed that P2X4 was required for the expression of retinoic acid-related orphan receptor C, which is the master regulator of Th17 cells. In contrast, inhibition of P2X4 receptor had no effect on Th1 cells and on the production of IFN-γ and it did not affect the expression of the transcription factor T-bet (T-box transcription factor). Furthermore, inhibition of P2X4 receptor reduced the production of IL-17 but not of IFN-γ by effector/memory CD4+ T cells isolated from patients with rheumatoid arthritis. In contrast to P2X4, inhibition of P2X7 and P2Y11 receptors had no effects on Th17 and Th1 cell activation. Finally, treatment with the P2X4 receptor antagonist 5-BDBD reduced the severity of collagen-induced arthritis in mice by inhibiting Th17 cell expansion and activation. Our findings provide novel insights into the role of purinergic signaling in T cell activation and identify a critical role for the purinergic receptor P2X4 in Th17 activation and in autoimmune arthritis.

Improvement of the affinity of an anti-rat P2X4 receptor antibody by introducing electrostatic interactions.

We have recently developed a mouse monoclonal antibody (12-10H) binding to the head domain region in rat P2X4 receptor (rP2X4R, which is crucial for the pathogenesis of neuropathic pain) expressed on the cell with the highest binding affinity (KD = 20 nM). However, the 12-10H antibody failed to detect endogenously expressed P2X4Rs in microglia isolated from the spinal cord of rats whose spinal nerves were injured. Then, we prepared R5 mutant, in which five arginine residues were introduced into variable regions except for the "hot spot" in the 12-10H antibody to increase electrostatic interactions with the head domain, an anionic region, in rP2X4R. The mutation resulted in an increase of 50-fold in the affinity of the R5 mutant for the head domain with respect to the intact 12-10H antibody. As a result, detection of P2X4Rs endogenously expressed on primary cultured microglial cells originated from the neonatal rat brain and spinal cord microglia isolated from a rat model of neuropathic pain was achieved. These findings suggest a strategy to improve the affinity of a monoclonal antibody for an anionic antigen by the introduction of several arginine residues into variable regions other than the "hot spot" in the paratope.

P2X4 purinergic receptors offer a therapeutic target for aggressive prostate cancer.

Prostate cancer (PCa) remains a leading cause of cancer-related deaths in American men and treatment options for metastatic PCa are limited. There is a critical need to identify new mechanisms that contribute to PCa progression, that distinguish benign from lethal disease, and that have potential for therapeutic targeting. P2X4 belongs to the P2 purinergic receptor family that is commonly upregulated in cancer and is associated with poorer outcomes. We observed P2X4 protein expression primarily in epithelial cells of the prostate, a subset of CD66+ neutrophils, and most CD68+ macrophages. Our analysis of tissue microarrays representing 491 PCa cases demonstrated significantly elevated P2X4 expression in cancer- compared with benign-tissue spots, in prostatic intraepithelial neoplasia, and in PCa with ERG positivity or with PTEN loss. High-level P2X4 expression in benign tissues was likewise associated with the development of metastasis after radical prostatectomy. Treatment with the P2X4-specific agonist cytidine 5'-triphosphate (CTP) increased Transwell migration and invasion of PC3, DU145, and CWR22Rv1 PCa cells. The P2X4 antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) resulted in a dose-dependent decrease in viability of PC3, DU145, LNCaP, CWR22Rv1, TRAMP-C2, Myc-CaP, BMPC1, and BMPC2 cells and decreased DU145 cell migration and invasion. Knockdown of P2X4 attenuated growth, migration, and invasion of PCa cells. Finally, knockdown of P2X4 in Myc-CaP cells resulted in significantly attenuated subcutaneous allograft growth in FVB/NJ mice. Collectively, these data strongly support a role for the P2X4 purinergic receptor in PCa aggressiveness and identify P2X4 as a candidate for therapeutic targeting. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Catestatin enhances ATP-induced activation of glial cells mediated by purinergic receptor P2X4.

The activation of glial cells and its possible mechanism play an extremely important role in understanding the pathophysiological process of some clinical diseases, and catestatin (CST) is involved in regulating this activation. In this project, we found that CST could enhance the activation of satellite glial cells (SGCs) and microglial cells and that the expression of P2X4 was increased; the co-expression of the P2X4 receptor with glial fibrillary acidic protein (GFAP) and the P2X4 receptor with CD11b was also increased significantly in glial cells of the ATP + CST group, and TNF-α and IL-1ß also showed a rising trend; the expression of phosphorylated ERK1/2 was also increased in the ATP + CST group. In summary, we conclude that CST could enhance ATP-induced activation of SGCs and microglial cells mediated by the P2X4 receptor and that the ERK1/2 signaling pathway may be involved in this activation process.

P2X4 Receptors Mediate Ca2+ Release from Lysosomes in Response to Stimulation of P2X7 and H1 Histamine Receptors.

The P2X4 purinergic receptor is targeted to endolysosomes, where it mediates an inward current dependent on luminal ATP and pH. Activation of P2X4 receptors was previously shown to trigger lysosome fusion, but the regulation of P2X4 receptors and their role in lysosomal Ca2+ signaling are poorly understood. We show that lysosomal P2X4 receptors are activated downstream of plasma membrane P2X7 and H1 histamine receptor stimulation. When P2X4 receptors are expressed, the increase in near-lysosome cytosolic [Ca2+] is exaggerated, as detected with a low-affinity targeted Ca2+ sensor. P2X4-dependent changes in lysosome properties were triggered downstream of P2X7 receptor activation, including an enlargement of lysosomes indicative of homotypic fusion and a redistribution of lysosomes towards the periphery of the cell. Lysosomal P2X4 receptors, therefore, have a role in regulating lysosomal Ca2+ release and the regulation of lysosomal membrane trafficking.