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The symptoms of fragile X syndrome (FXS), caused by a single gene mutation to Fmr1, have been increasingly linked to disordered astrocyte signalling within the cerebral cortex. We have recently demonstrated that the purinergic signalling pathway, which utilizes nucleoside triphosphates and their metabolites to facilitate bidirectional glial and glial-neuronal interactions, is upregulated in cortical astrocytes derived from the Fmr1 knockout (KO) mouse model of FXS. Heightened Fmr1 KO P2Y purinergic receptor levels were correlated with prolonged intracellular calcium release, elevated synaptogenic protein secretion, and hyperactivity of developing circuits. However, due to the relative lack of sensitive and reproducible quantification methods available for measuring purines and pyrimidines, determining the abundance of these factors in Fmr1 KO astrocytes was limited. We therefore developed a hydrophilic interaction liquid chromatography protocol coupled with mass spectrometry to compare the abundance of intracellular and extracellular purinergic molecules between wildtype and Fmr1 KO mouse astrocytes. Significant differences in the concentrations of UDP, ATP, AMP, and adenosine intracellular stores were found within Fmr1 KO astrocytes relative to WT. The extracellular level of adenosine was also significantly elevated in Fmr1 KO astrocyte-conditioned media in comparison to media collected from WT astrocytes. Glycosylation of the astrocyte membrane-bound CD39 ectonucleotidase, which facilitates ligand breakdown following synaptic release, was also elevated in Fmr1 KO astrocyte cultures. Together, these differences demonstrated further dysregulation of the purinergic signalling system within Fmr1 KO cortical astrocytes, potentially leading to significant alterations in FXS purinergic receptor activation and cellular pathology., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: CRLF2 alterations are associated with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This study aimed to explore the clinical, biological, and outcome features of pediatric BCP-ALL with CRLF2 abnormalities., Methods: This study enrolled 630 childhood BCP-ALLs treated on CCLG-ALL 2008 or 2018 protocol. P2RY8-CRLF2 was determined by Sanger sequencing and CRLF2 expression was evaluated by qRT-PCR. The correlation between clinical, biological features and outcomes with P2RY8-CRLF2 or CRLF2 over-expression were analyzed., Results: P2RY8-CRLF2 and CRLF2 over-expression were found in 3.33% and 5.71% respectively. P2RY8-CRLF2 was associated with male, higher frequency of CD7 expression, high WBC and MRD before consolidation. CRLF2 over-expression showed ETV6-RUNX1 - , higher frequency of CD22, CD34, CD66c, CD86 expression, hyperdiploidy and high MRD at early treatment. The lower overall survival (OS) was found in patients with P2RY8-CRLF2 and confined only in IR group. Furthermore, adverse event-free survival and OS of P2RY8-CRLF2 were discovered comparing to those without known fusions or treated on CCLG-ALL 2008 protocol. However, P2RY8-CRLF2 was not confirmed as independent prognostic factors and no prognostic impact of CRLF2 over-expression was found., Conclusions: These findings indicate P2RY8-CRLF2 identifies a subset of patients with specific features and adverse outcomes that could be improved by risk-directed treatment., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

B cell self-tolerance is maintained through multiple checkpoints, including restraints on intracellular signaling and cell trafficking. P2RY8 is a receptor with established roles in germinal center (GC) B cell migration inhibition and growth regulation. Somatic P2RY8 variants are common in GC-derived B cell lymphomas. Here, we identify germline novel or rare P2RY8 missense variants in lupus kindreds or the related antiphospholipid syndrome, including a "de novo" variant in a child with severe nephritis. All variants decreased protein expression, F-actin abundance, and GPCR-RhoA signaling, and those with stronger effects increased AKT and ERK activity and cell migration. Remarkably, P2RY8 was reduced in B cell subsets from some SLE patients lacking P2RY8 gene variants. Low P2RY8 correlated with lupus nephritis and increased age-associated B cells and plasma cells. By contrast, P2RY8 overexpression in cells and mice restrained plasma cell development and reinforced negative selection of DNA-reactive developing B cells. These findings uncover a role of P2RY8 in immunological tolerance and lupus pathogenesis., Competing Interests: Disclosures:  J.G. Cyster reported "other" from Be BioPharma and MiroBio outside the submitted work. No other disclosures were reported., (© 2021 He et al.)

G-protein-coupled receptors (GPCRs), especially chemokine receptors, play a central role in the regulation of T cell migration. Various GPCRs are upregulated in activated CD4 T cells, including P2Y10, a putative lysophospholipid receptor that is officially still considered an orphan GPCR, i.e., a receptor with unknown endogenous ligand. Here we show that in mice lacking P2Y10 in the CD4 T cell compartment, the severity of experimental autoimmune encephalomyelitis and cutaneous contact hypersensitivity is reduced. P2Y10-deficient CD4 T cells show normal activation, proliferation and differentiation, but reduced chemokine-induced migration, polarization, and RhoA activation upon in vitro stimulation. Mechanistically, CD4 T cells release the putative P2Y10 ligands lysophosphatidylserine and ATP upon chemokine exposure, and these mediators induce P2Y10-dependent RhoA activation in an autocrine/paracrine fashion. ATP degradation impairs RhoA activation and migration in control CD4 T cells, but not in P2Y10-deficient CD4 T cells. Importantly, the P2Y10 pathway appears to be conserved in human T cells. Taken together, P2Y10 mediates RhoA activation in CD4 T cells in response to auto-/paracrine-acting mediators such as LysoPS and ATP, thereby facilitating chemokine-induced migration and, consecutively, T cell-mediated diseases., (© 2021. The Author(s).)

P2RY8 promotes the confinement and growth regulation of germinal center (GC) B cells, and loss of human P2RY8 is associated with B cell lymphomagenesis. The metabolite S -geranylgeranyl-l-glutathione (GGG) is a P2RY8 ligand. The mechanisms controlling GGG distribution are poorly understood. Here, we show that gamma-glutamyltransferase-5 (Ggt5) expression in stromal cells was required for GGG catabolism and confinement of P2RY8-expressing cells to GCs. We identified the ATP-binding cassette subfamily C member 1 (Abcc1) as a GGG transporter and showed that Abcc1 expression by hematopoietic cells was necessary for P2RY8-mediated GC confinement. Furthermore, we discovered that P2RY8 and GGG negatively regulated trafficking of B and T cells to the bone marrow (BM). P2RY8 loss-of-function human T cells increased their BM homing. By defining how GGG distribution was determined and identifying sites of P2RY8 activity, this work helps establish how disruptions in P2RY8 function contribute to lymphomagenesis and other disease states., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Objective: To investigate the clinical features and prognostic factors of acute lymphoblastic leukemia (ALL) children with P2RY8-CRLF2 gene rearrangement., Methods: A total of 108 children with B-cell ALL (B-ALL) were diagnosed and systematically treated according to Chinese Children's Leukemia Group (CCLG) -ALL 2008 in our hospital from January 2016 to December 2016. The 108 patients were divided into two groups according to the result of mutiplex polymerase chain reaction: group with P2RY8-CRLF2 gene rearrangement and group without P2RY8-CRLF2 gene rearrangement. The ALL children with P2RY8-CRLF2 gene rearrangement were all treated by CCLG-ALL 2008 high-risk group (HR) regimens, and the ALL children in group without P2RY8-CRLF2 gene rearrangement received different intensity chemotherapy according to clinical risk classification., Results: Five (4 male and 1 female) out of 108 patients with B-ALL had P2RY8-CRLF2 gene rearrangement. In the 5 B-ALL patients with P2RY8-CRLF2 gene rearrangement, the median age of the was 4 (2-6) years old and the median WBC count was 26.2 (2.46-525.1)×10 9 /L. These patients presented different immunophenotype, including 3 cases of common B-ALL and 2 cases of pre B-ALL. Four patients carried a normal karyotype and 1 patient carried 46, XY, der (20) [22]/46, XY[2]. For the children with P2RY8-CRLF2 gene rearrangement, 1 patient (20%) could not achieve complete remission (CR), and minimal residual disease (MRD) of 2 patients (40%) was higher than 1% on day 33 of induction chemotherapy; while in group without P2RY8-CRLF2 gene rearrangement, all the patient achieved CR, and MRD in 6 patients (5.8%) was higher than 1% on day 33 of induction chemotherapy. The 3 year event-free survival (EFS) of ALL children in group with P2RY8-CRLF2 gene rearrangement was significantly lower than that in group without P2RY8-CRLF2 gene rearrangement (60.0%±21.9% vs 85.9%±3.9%) (P<0.05)., Conclusion: The early treatment response and prognosis of ALL children with P2RY8-CRLF2 gene rearrangement are worse, and more effective protocol is needed for this subtype patients.

Accumulating evidence has shown that long non‑coding RNAs (lncRNAs) play significant roles in the development and progression of many types of cancer including colorectal cancer. RP11‑400N13.3 is a novel lncRNA discovered recently and its biological function and underlying mechanism in colorectal cancer remain elusive. This study aimed to reveal the relationship between RP11‑400N13.3 and colorectal cancer. Our results demonstrated that the expression of RP11‑400N13.3 was significantly upregulated in both colorectal cancer tissues and cell lines as compared to normal adjacent tissues and normal colonic epithelial cells by RT‑qPCR, respectively. Upregulation of RP11‑400N13.3 was found to be correlated with a poor overall survival rate. Functional studies revealed that RP11‑400N13.3 facilitated the proliferation, migration, invasion and tumor growth of colorectal cancer cells while inhibiting the apoptosis of cancer cells in vitro and in vivo. We also observed that RP11‑400N13.3 serves as a sponge for miR‑4722‑3p, and that P2Y receptor family member 8 (P2RY8) was predicted to be a target of miR‑4722‑3p by bioinformatics analysis. Western blot assay indicated that the expression of P2RY8 was negatively or positively regulated by miR‑4722‑3p or RP11‑400N13.3. In addition, rescue experiments revealed that RP11‑400N13.3 promoted proliferation, migration and invasion by directly regulating the expression of miR‑4722‑3p and P2RY8. In conclusion, our results revealed that RP11‑400N13.3 promoted colorectal cancer progression via modulating the miR‑4722‑3p/P2RY8 axis, thus suggesting RP11‑400N13.3 as a potential therapeutic target for the treatment of colorectal cancer.

The release and extracellular action of ATP are a widespread mechanism for cell-to-cell communication in living organisms through activation of P2X and P2Y receptors expressed at the cell surface of most tissues, including the nervous system. Among ionototropic receptors, P2X4 receptors have emerged in the last decade as a potential target for CNS disorders such as epilepsy, ischemia, chronic pain, anxiety, multiple sclerosis and neurodegenerative diseases. However, the role of P2X4 receptor in each pathology ranges from beneficial to detrimental, although the mechanisms are still mostly unknown. P2X4 is expressed at low levels in CNS cells including neurons and glial cells. In normal conditions, P2X4 activation contributes to synaptic transmission and synaptic plasticity. Importantly, one of the genes present in the transcriptional program of myeloid cell activation is P2X4. Microglial P2X4 upregulation, the P2X4 + state of microglia, seems to be common in most acute and chronic neurodegenerative diseases associated with inflammation. In this review, we summarize knowledge about the role of P2X4 receptors in the CNS physiology and discuss potential pitfalls and open questions about the therapeutic potential of blocking or potentiation of P2X4 for different pathologies.

Ischemic acute kidney injury (AKI), a complication that frequently occurs in hospital settings, is often associated with hemodynamic compromise, sepsis, cardiac surgery, or exposure to nephrotoxins. Here, using a murine renal ischemia/reperfusion injury (IRI) model, we show that intercalated cells (ICs) rapidly adopted a proinflammatory phenotype after IRI. Wwe demonstrate that during the early phase of AKI either blockade of the proinflammatory P2Y14 receptor located on the apical membrane of ICs or ablation of the gene encoding the P2Y14 receptor in ICs (a) inhibited IRI-induced increase of chemokine expression in ICs, (b) reduced neutrophil and monocyte renal infiltration, (c) reduced the extent of kidney dysfunction, and (d) attenuated proximal tubule damage. These observations indicate that the P2Y14 receptor participates in the very first inflammatory steps associated with ischemic AKI. In addition, we show that the concentration of the P2Y14 receptor ligand UDP-glucose (UDP-Glc) was higher in urine samples from intensive care unit patients who developed AKI compared with patients without AKI. In particular, we observed a strong correlation between UDP-Glc concentration and the development of AKI in cardiac surgery patients. Our study identifies the UDP-Glc/P2Y14 receptor axis as a potential target for the prevention and/or attenuation of ischemic AKI.

The purinergic (P2) receptor P2Y 14 is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y 14 expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). UDP-glucose (UDPG) had undiscernible effects on cAMP levels, however, induced dose-dependent elevations in the cytosolic free calcium concentration ([Ca 2+ ] i ) in CB-Ob, but not C2-Ob cells. To antagonize the P2Y 14 function, we used the P2Y 14 inhibitor PPTN or generated CRISPR-Cas9-mediated P2Y 14 knockout C2-Ob clones (Y14 KO ). P2Y 14 inhibition facilitated calcium signalling and altered basal cAMP levels in both models of osteoblasts. Importantly, P2Y 14 inhibition augmented Ca 2+ signalling in response to ATP, ADP and mechanical stimulation. P2Y 14 knockout or inhibition reduced osteoblast proliferation and decreased ERK1/2 phosphorylation and increased AMPKα phosphorylation. During in vitro osteogenic differentiation, P2Y 14 inhibition modulated the timing of osteogenic gene expression, collagen deposition, and mineralization, but did not significantly affect differentiation status by day 28. Of interest, while P2ry14 -/- mice from the International Mouse Phenotyping Consortium were similar to wild-type controls in bone mineral density, their tibia length was significantly increased. We conclude that P2Y 14 in osteoblasts reduces cell responsiveness to mechanical stimulation and mechanotransductive signalling and modulates osteoblast differentiation.

Extracellular ATP through the activation of the P2X and P2Y purinergic receptors affects the migration, proliferation and differentiation of many types of cells, including stem cells. High plasticity, low immunogenicity and immunomodulation ability of mesenchymal stem cells derived from human endometrium (eMSCs) allow them to be considered a prominent tool for regenerative medicine. Here, we examined the role of ATP in the proliferation and migration of human eMSCs. Using a wound healing assay, we showed that ATP-induced activation of purinergic receptors suppressed the migration ability of eMSCs. We found the expression of one of the ATP receptors, the P2X 7 receptor in eMSCs. In spite of this, cell activation with specific P2X 7 receptor agonist, BzATP did not significantly affect the cell migration. The allosteric P2X 7 receptor inhibitor, AZ10606120 also did not prevent ATP-induced inhibition of cell migration, confirming that inhibition occurs without P2X 7 receptor involvement. Flow cytometry analysis showed that high concentrations of ATP did not have a cytotoxic effect on eMSCs. At the same time, ATP induced the cell cycle arrest, suppressed the proliferative and migration capacity of eMSCs and therefore could affect the regenerative potential of these cells., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Angiogenic vasa vasorum (VV) expansion plays an essential role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH), a cardiovascular disease. We previously showed that extracellular ATP released under hypoxic conditions is an autocrine/paracrine, the angiogenic factor for pulmonary artery (PA) VV endothelial cells (VVECs), acting via P2Y purinergic receptors (P2YR) and the Phosphoinositide 3-kinase (PI3K)-Akt-Mammalian Target of Rapamycin (mTOR) signaling. To further elucidate the molecular mechanisms of ATP-mediated VV angiogenesis, we determined the profile of ATP-inducible transcription factors (TFs) in VVECs using a TranSignal protein/DNA array. C-Jun, c-Myc, and Foxo3 were found to be upregulated in most VVEC populations and formed nodes connecting several signaling networks. siRNA-mediated knockdown (KD) of these TFs revealed their critical role in ATP-induced VVEC angiogenic responses and the regulation of downstream targets involved in tissue remodeling, cell cycle control, expression of endothelial markers, cell adhesion, and junction proteins. Our results showed that c-Jun was required for the expression of ATP-stimulated angiogenic genes, c-Myc was repressive to anti-angiogenic genes, and Foxo3a predominantly controlled the expression of anti-apoptotic and junctional proteins. The findings from our study suggest that pharmacological targeting of the components of P2YR-PI3K-Akt-mTOR axis and specific TFs reduced ATP-mediated VVEC angiogenic response and may have a potential translational significance in attenuating pathological vascular remodeling., Competing Interests: The authors declare no conflicts of interest.

P2Y purinergic receptors expressed in neurons and satellite glial cells (SGCs) of the trigeminal ganglion (TG) contribute to inflammatory and neuropathic pain. P2Y 14 receptor expression is reported in the spinal cord, dorsal root ganglion (DRG), and TG. In present study, the role of P2Y 14 receptor in the TG in inflammatory orofacial pain of Sprague-Dawley (SD) rats was investigated. Peripheral injection of complete Freund's adjuvant (CFA) induced mechanical hyperalgesia with the rapid upregulation of P2Y 14 receptor, glial fibrillary acidic protein (GFAP), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), C-C chemokine CCL2, phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), and phosphorylated p38 (p-p38) proteins in the TG. Furthermore, immunofluorescence staining confirmed the CFA-induced upregulation of P2Y 14 receptor. Double immunostaining showed that P2Y 14 receptor colocalized with glutamine synthetase (GS) and neuronal nuclei (NeuN). Finally, trigeminal injection of a selective antagonist (PPTN) of P2Y 14 receptor attenuated CFA-induced mechanical hyperalgesia. PPTN also decreased the upregulation of the GFAP, IL-1β, TNF-α, CCL2, p-ERK1/2, and p-p38 proteins. Our findings showed that P2Y 14 receptor in TG may contribute to orofacial inflammatory pain via regulating SGCs activation, releasing cytokines (IL-1β, TNF-α, and CCL2), and phosphorylating ERK1/2 and p38., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Non-neuronal ATP released from the urothelium in response to bladder stretch is a key modulator of bladder mechanosensation. Whilst non-neuronal ATP acts on the underlying bladder afferent nerves to facilitate sensation, there is also the potential for ATP to act in an autocrine manner, modulating urothelial cell function. The aim of this study was to systematically characterise the functional response of primary mouse urothelial cells (PMUCs) to ATP. PMUCs isolated from male mice (14-16 weeks) were used for live-cell fluorescent calcium imaging and qRT-PCR to determine the expression profile of P2X and P2Y receptors. The majority of PMUCs (74-92%) responded to ATP (1 μM-1 mM), as indicted by an increase in intracellular calcium (iCa 2+ ). PMUCs exhibited dose-dependent responses to ATP (10 nM-1 mM) in both calcium containing (2 mM, EC 50  = 3.49 ± 0.77 μM) or calcium free (0 mM, EC 50  = 9.5 ± 1.5 μM) buffers. However, maximum iCa 2+ responses to ATP were significantly attenuated upon repetitive applications in calcium containing but not in calcium free buffer. qRT-PCR revealed expression of P2X 1-6 , and P2Y 1-2 , P2Y 4 , P2Y 6 , P2Y 11-14 , but not P2X 7 in PMUCs. These findings suggest the major component of ATP induced increases in iCa 2+ are mediated via the liberation of calcium from intracellular stores, implicating functional P2Y receptors that are ubiquitously expressed on PMUCs.

Extracellular nucleotides and nucleotide sugars are important danger-associated signaling molecules that play critical roles in regulation of immune responses in mammals through activation of purinergic receptors located on the cell surface. However, the immunological role of extracellular UDP-glucose-activated P2Y 14 receptor (P2Y 14 R) in fish still remains unknown. In this study, we identified and characterized a P2Y 14 R paralog in the Japanese flounder (Paralichthys olivaceus). The mRNA transcripts of P2Y 14 R are detected in all examined Japanese flounder tissues. Compared with the UDP-activated P2Y 6 receptor, however, P2Y 14 R gene is highly expressed in Japanese flounder head kidney macrophages (HKMs). In addition, P2Y 14 R is significantly upregulated following inflammatory stimulation with LPS and poly (I:C) in the HKMs, suggesting a role of P2Y 14 R in response to inflammation in fish. Furthermore, activation of P2Y 14 receptor with its potent and selective agonist MRS 2905 resulted in a decreased expression of LPS-induced pro-inflammatory cytokine IL-1beta gene in the HKMs. In contrast, inhibition of P2Y 14 receptor activity or down-regulation of the endogenous expression of P2Y 14 R by small interfering RNA significantly upregulates the LPS-induced pro-inflammatory cytokine IL-1beta gene expression in the HKMs, demonstrating that P2Y 14 R is involved in inflammation regulation in fish. Moreover, stimulation of the Japanese flounder HKMs with UDP-glucose evoked a rapid increase of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in a dose- and time-dependent manner, indicating the involvement of P2Y 14 R in activation of ERK1/2 signaling in fish immune cells. Taken together, we demonstrated that the inducible P2Y 14 R plays an important role in regulation of fish innate immunity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The germline variant at rs3824662 in GATA3 is a risk locus for Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), the biological subtype of B-cell precursor (BCP)-ALL defined by a distinct gene expression profile and the presence of specific somatic aberrations including rearrangements of CRLF2. In this study, we investigated whether rs3824662 in GATA3 associates with CRLF2 expression in leukemic cells and predicts prognosis in pediatric BCP-ALL patients treated according to the ALL Intercontinental Berlin-Frankfurt-Münster (IC BFM) 2009 (n = 645) and the ALL IC BFM 2002 (n = 216) protocols. High expression of CRLF2 was observed at both protein and mRNA levels (fourfold higher in AA than in CA + CC) among GATA3 AA variant carriers, independent of the presence of P2RY8-CRLF2 fusion. Additionally, the AA variant at rs3824662 was a significant factor affecting minimal residual disease level at the end of induction phase and overall survival regardless of the risk group and the protocol. The germline variant at rs3824662 in GATA3 is a prognostic factor which associates with CRLF2 expression in leukemic cells supporting the hypothesis that GATA3 may have a regulatory effect on the CRLF2 pathway in pediatric BCP-ALL., (© 2019 Wiley Periodicals, Inc.)

Germinal centres are important sites for antibody diversification and affinity maturation, and are also a common origin of B cell malignancies. Despite being made up of motile cells, germinal centres are tightly confined within B cell follicles. The cues that promote this confinement are incompletely understood. P2RY8 is a Gα 13 -coupled receptor that mediates the inhibition of migration and regulates the growth of B cells in lymphoid tissues 1,2 . P2RY8 is frequently mutated in germinal-centre B cell-like diffuse large B cell lymphoma (GCB-DLBCL) and Burkitt lymphoma 1,3-6 , and the ligand for this receptor has not yet been identified. Here we perform a search for P2RY8 ligands and find P2RY8 bioactivity in bile and in culture supernatants of several mouse and human cell lines. Using a seven-step biochemical fractionation procedure and a drop-out mass spectrometry approach, we show that a previously undescribed biomolecule, S-geranylgeranyl-L-glutathione (GGG), is a potent P2RY8 ligand that is detectable in lymphoid tissues at the nanomolar level. GGG inhibited the chemokine-mediated migration of human germinal-centre B cells and T follicular helper cells, and antagonized the induction of phosphorylated AKT in germinal-centre B cells. We also found that the enzyme gamma-glutamyltransferase-5 (GGT5), which was highly expressed by follicular dendritic cells, metabolized GGG to a form that did not activate the receptor. Overexpression of GGT5 disrupted the ability of P2RY8 to promote B cell confinement to germinal centres, which indicates that GGT5 establishes a GGG gradient in lymphoid tissues. This work defines GGG as an intercellular signalling molecule that is involved in organizing and controlling germinal-centre responses. As the P2RY8 locus is modified in several other types of cancer in addition to GCB-DLBCL and Burkitt lymphoma, we speculate that GGG might have organizing and growth-regulatory roles in multiple human tissues.

Background: Genomic profiling efforts have revealed a rich diversity of oncogenic fusion genes. While there are many methods for identifying fusion genes from RNA-sequencing (RNA-seq) data, visualizing these transcripts and their supporting reads remains challenging., Findings: Clinker is a bioinformatics tool written in Python, R, and Bpipe that leverages the superTranscript method to visualize fusion genes. We demonstrate the use of Clinker to obtain interpretable visualizations of the RNA-seq data that lead to fusion calls. In addition, we use Clinker to explore multiple fusion transcripts with novel breakpoints within the P2RY8-CRLF2 fusion gene in B-cell acute lymphoblastic leukemia., Conclusions: Clinker is freely available software that allows visualization of fusion genes and the RNA-seq data used in their discovery.

The PI3K/mTOR pathway is the second most frequently deregulated pathway in a majority of cancers such as breast cancer, lung cancer, and melanomas as well as leukemia. Mutations in the genes coding for receptor tyrosine kinases (RTKs) and G-protein-coupled receptors (GPCRs) are quite common in all forms of acute leukemia. This can be a major cause of deregulation of the PI3K-mTOR pathway. To understand how cells display resistance to the dual PI3K/mTOR inhibitor, we used a panel of 25 acute leukemia cell lines. We observed that while a number of cell lines displayed sensitivity to the dual PI3K/mTOR pathway inhibitor PKI-587, many cells displayed substantial resistance. Cells sensitive to PKI-587 also showed aberrant activation of PI3K/mTOR pathway components such as AKT and S6K and also displayed sensitivity to a panel of various other PI3K/mTOR inhibitors. Using RNA sequencing data, we observed that expression of a G protein-coupled receptor, P2RY14, was upregulated nine-fold in cells showing resistance to the PI3K/mTOR inhibitor. P2RY14 has not been much studied in hematologic malignancies. However, this receptor seems to have a role in the localization of hematopoietic stem cells (HSCs) and in promoting regenerative capabilities following injury. We observed that acute lymphoblastic leukemia (ALL) and FLT3-ITD-positive acute myeloid leukemia (AML) patients with higher expression of P2RY14 mRNA displayed relatively poor survival compared to patients carrying lower expression of P2RY14 suggesting a role of P2RY14 in patient survival. To understand the role of this receptor in cell signaling, we used phospho-protein arrays and observed activation of distinct signaling cascades. Furthermore, array data were verified using murine pro-B cell line Ba/F3 stably transfected with P2RY14. We observed that activation of P2RY14 by its ligand, UDP-glucose, resulted in selective induction of ERK1/2 phosphorylation. Taken together, our data suggest that acute leukemia cells resistant to PI3K/mTOR inhibition display upregulation of a GPCR, P2RY14, which has a role in patient survival and also couples to the activation of ERK signaling., Competing Interests: Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

The purinergic transduction was examined in mesenchymal stromal cells (MSCs) from the human adipose tissue, and several nucleotides, including ATP, UTP, and ADP, were found to mobilize cytosolic Ca 2+ . Transcripts for multiple purinoreceptors were detected in MSC preparations, including A 1 , A 2A , A 2B , P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 13 , P2Y 14 , P2X 2 , P2X 4 , and P2X 7 . Cellular responses to nucleotides were insignificantly sensitive to bath Ca 2+ , pointing at a minor contribution of Ca 2+ entry, and were suppressed by U73122 and 2-APB, implicating the phosphoinositide cascade in coupling P2Y receptors to Ca 2+ release. While individual cells were sensitive to several P2Y agonists, responsiveness to a given nucleotide varied from cell to cell, suggesting that particular MSCs could employ different sets of purinoreceptors. Caged Ca 2+ stimulated Ca 2+ -induced Ca 2+ release (CICR) that was mediated largely by IP 3 receptors, and resultant Ca 2+ transients were similar to nucleotide responses by magnitude and kinetics. A variety of findings hinted at CICR to be a universal mechanism that finalizes Ca 2+ signaling initiated by agonists in MSCs. Individual MSCs responded to nucleotides in an all-or-nothing manner. Presumably just CICR provided invariant Ca 2+ responses observed in MSCs at different nucleotide concentrations. The effects of isoform specific agonists and antagonists suggested that both P2Y 1 and P2Y 13 were obligatory for ADP responses, while P2Y 4 and P2Y 11 served as primary UTP and ATP receptors, respectively. Extracellular NAD + stimulated Ca 2+ signaling in each ATP-responsive MSC by involving P2Y 11 . The overall data indicate that extracellular nucleotides and NAD + can serve as autocrine/paracrine factors regulating MSC functions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This review is focused on the pathophysiology and therapeutic potential of purinergic signalling. A wide range of diseases are considered, including those of the central nervous system, skin, kidney, musculoskeletal, liver gut, lower urinary tract, cardiovascular, airways and reproductive systems, the special senses, infection, diabetes and obesity. Several purinergic drugs are already on the market, including P2Y 12 receptor antagonists for stroke and thrombosis, P2Y 2 receptor agonists for dry eye, and A 1 receptor agonists for supraventricular tachycardia. Clinical trials are underway for the use of P2X3 receptor antagonists for the treatment of chronic cough, visceral pain and hypertension, and many more compounds are being explored for the treatment of other diseases. Most experiments are 'proof of concept' studies on animal or cellular models, which hopefully will lead to further clinical trials. The review summarises the topic, mostly referring to recent review articles., (Copyright © 2017 Elsevier Inc. All rights reserved.)

While interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine essential for host defense, high systemic levels cause life-threatening inflammatory syndromes. ATP, a stimulus of IL-1β maturation, is released from damaged cells along with β-nicotinamide adenine dinucleotide (β-NAD). Here, we tested the hypothesis that β-NAD controls ATP-signaling and, hence, IL-1β release. Lipopolysaccharide-primed monocytic U937 cells and primary human mononuclear leukocytes were stimulated with 2'(3')- O -(4-benzoyl-benzoyl)ATP trieethylammonium salt (BzATP), a P2X7 receptor agonist, in the presence or absence of β-NAD. IL-1β was measured in cell culture supernatants. The roles of P2Y receptors, nicotinic acetylcholine receptors (nAChRs), and Ca 2+ -independent phospholipase A2 (iPLA2β, PLA2G6) were investigated using specific inhibitors and gene-silencing. Exogenous β-NAD signaled via P2Y receptors and dose-dependently (IC 50 = 15 µM) suppressed the BzATP-induced IL-1β release. Signaling involved iPLA2β, release of a soluble mediator, and nAChR subunit α9. Patch-clamp experiments revealed that β-NAD inhibited BzATP-induced ion currents. In conclusion, we describe a novel triple membrane-passing signaling cascade triggered by extracellular β-NAD that suppresses ATP-induced release of IL-1β by monocytic cells. This cascade links activation of P2Y receptors to non-canonical metabotropic functions of nAChRs that inhibit P2X7 receptor function. The biomedical relevance of this mechanism might be the control of trauma-associated systemic inflammation., Competing Interests: J. Michael McIntosh is an inventor on patents regarding some nicotinic antagonists used in this study. All other authors declare no conflict of interest.

Background: A high-level expression of the CRLF2 gene is frequent in precursor B-cell acute lymphoblastic leukemia (pB-ALL) and can be caused by different genetic aberrations. The presence of the most frequent alteration, the P2RY8/CRLF2 fusion, was shown to be associated with a high relapse incidence in children treated according to ALL-Berlin-Frankfurt-Münster (BFM) protocols, which is poorly understood. Moreover, the frequency of other alterations has not been systematically analyzed yet., Procedure: CRLF2 mRNA expression and potential genetic aberrations causing a CRLF2 high expression were prospectively assessed in 1,105 patients treated according to the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP)-BFM ALL 2009 protocol. Additionally, we determined copy number alterations in selected B-cell differentiation genes for all CRLF2 high-expressing pB-ALL cases, as well as JAK2 and CRLF2 mutations., Results: A CRLF2 high expression was detected in 26/178 (15%) T-cell acute lymphoblastic leukemia (T-ALL) cases, 21 of them (81%) had been stratified as high-risk patients by treatment response. In pB-ALL, a CRLF2 high expression was determined in 91/927 (10%) cases; the P2RY8/CRLF2 rearrangement in 44/91 (48%) of them, supernumerary copies of CRLF2 in 18/91 (20%), and, notably, the IGH/CRLF2 translocation was detected in 16/91 (18%). Remarkably, 7 of 16 (44%) patients with IGH/CRLF2 translocation had already relapsed. P2RY8/CRLF2- and IGH/CRLF2-positive samples (70 and 94%, respectively) were characterized by a high frequency of additional deletions in B-cell differentiation genes such as IKZF1 or PAX5., Conclusion: Our data suggest that this high frequency of genetic aberrations in the context of a high CRLF2 expression could contribute to the high risk of relapse in P2RY8/CRLF2- and IGH/CRLF2-positive ALL., (© 2017 Wiley Periodicals, Inc.)

Children with P2RY8-CRLF2-positive acute lymphoblastic leukemia have an increased relapse risk. Their mutational and transcriptional landscape, as well as the respective patterns at relapse remain largely elusive. We, therefore, performed an integrated analysis of whole-exome and RNA sequencing in 41 major clone fusion-positive cases including 19 matched diagnosis/relapse pairs. We detected a variety of frequently subclonal and highly instable JAK/STAT but also RTK/Ras pathway-activating mutations in 76% of cases at diagnosis and virtually all relapses. Unlike P2RY8-CRLF2 that was lost in 32% of relapses, all other genomic alterations affecting lymphoid development (58%) and cell cycle (39%) remained stable. Only IKZF1 alterations predominated in relapsing cases (P=0.001) and increased from initially 36 to 58% in matched cases. IKZF1's critical role is further corroborated by its specific transcriptional signature comprising stem cell features with signs of impaired lymphoid differentiation, enhanced focal adhesion, activated hypoxia pathway, deregulated cell cycle and increased drug resistance. Our findings support the notion that P2RY8-CRLF2 is dispensable for relapse development and instead highlight the prominent rank of IKZF1 for relapse development by mediating self-renewal and homing to the bone marrow niche. Consequently, reverting aberrant IKAROS signaling or its disparate programs emerges as an attractive potential treatment option in these leukemias.

Epicardium-derived cells (EPDCs) play a fundamental role in embryonic cardiac development and are reactivated in the adult heart in response to myocardial infarction (MI). In this study, EPDCs from post-MI rat hearts highly expressed the ectoenzyme CD73 and secreted the profibrotic matricellular protein tenascin-C (TNC). CD73 on EPDCs extensively generated adenosine from both extracellular ATP and NAD. This in turn stimulated the release of additional nucleotides from a Brefeldin A-sensitive intracellular pool via adenosine-A 2B R signaling, forming a positive-feedback loop. A 2B R activation, in addition, strongly promoted the release of major regulatory cytokines, such as IL-6, IL-11, and VEGF. TNC was found to stimulate EPDC migration and, together with ATP-P2X 7 R signaling, to activate inflammasomes in EPDCs via TLR4. Our results demonstrate that EPDCs are an important source of various proinflammatory factors in the post-MI heart controlled by purinergic and TNC signaling.-Hesse, J., Leberling, S., Boden, E., Friebe, D., Schmidt, T., Ding, Z., Dieterich, P., Deussen, A., Roderigo, C., Rose, C. R., Floss, D. M., Scheller, J., Schrader, J. CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction., (© FASEB.)

Extracellular adenosine 5'-triphosphate (ATP) and adenosine molecules are intimately involved in immune responses. ATP is mostly a pro-inflammatory molecule and is released during hypoxic condition and by necrotic cells, as well as by activated immune cells and endothelial cells. However, under certain conditions, for instance at low concentrations or at prolonged exposure, ATP may also have anti-inflammatory properties. Extracellular ATP can activate both P2X and P2Y purinergic receptors. Extracellular ATP can be hydrolyzed into adenosine in a two-step enzymatic process involving the ectonucleotidases CD39 (ecto-apyrase) and CD73. These enzymes are expressed by many cell types, including endothelial cells and immune cells. The counterpart of ATP is adenosine, which is produced by breakdown of intra- or extracellular ATP. Adenosine has mainly anti-inflammatory effects by binding to the adenosine, or P1, receptors (A1, A2A, A2B, and A3). These receptors are also expressed in many cells, including immune cells. The final effect of ATP and adenosine in immune responses depends on the fine regulatory balance between the 2 molecules. In the present review, we will discuss the current knowledge on the role of these 2 molecules in the immune responses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The β 2 adrenergic receptor ( β 2 AR) increases intracellular Ca 2+ in a variety of cell types. By combining pharmacological and genetic manipulations, we reveal a novel mechanism through which the β 2 AR promotes Ca 2+ mobilization (pEC 50 = 7.32 ± 0.10) in nonexcitable human embryonic kidney (HEK)293S cells. Downregulation of Gs with sustained cholera toxin pretreatment and the use of Gs-null HEK293 (∆Gs-HEK293) cells generated using the clustered regularly interspaced short palindromic repeat-associated protein-9 nuclease (CRISPR/Cas9) system, combined with pharmacological modulation of cAMP formation, revealed a Gs-dependent but cAMP-independent increase in intracellular Ca 2+ following β 2 AR stimulation. The increase in cytoplasmic Ca 2+ was inhibited by P2Y purinergic receptor antagonists as well as a dominant-negative mutant form of Gq, a Gq-selective inhibitor, and an inositol 1,4,5-trisphosphate (IP 3 ) receptor antagonist, suggesting a role for this Gq-coupled receptor family downstream of the β 2 AR activation. Consistent with this mechanism, β 2 AR stimulation promoted the extracellular release of ATP, and pretreatment with apyrase inhibited the β 2 AR-promoted Ca 2+ mobilization. Together, these data support a model whereby the β 2 AR stimulates a Gs-dependent release of ATP, which transactivates Gq-coupled P2Y receptors through an inside-out mechanism, leading to a Gq- and IP 3 -dependent Ca 2+ mobilization from intracellular stores. Given that β 2 AR and P2Y receptors are coexpressed in various tissues, this novel signaling paradigm could be physiologically important and have therapeutic implications. In addition, this study reports the generation and validation of HEK293 cells deleted of Gs using the CRISPR/Cas9 genome editing technology that will undoubtedly be powerful tools to study Gs-dependent signaling., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Hundreds of genetic variants are thought to contribute to variation in asthma risk by modulating gene expression. Methods that increase the power of genome-wide association studies (GWASs) to identify risk-associated variants are needed., Objective: We sought to develop a method that aggregates the evidence for association with disease risk across expression quantitative trait loci (eQTLs) of a gene and use this approach to identify asthma risk genes., Methods: We developed a gene-based test and software package called EUGENE that (1) is applicable to GWAS summary statistics; (2) considers both cis- and trans-eQTLs; (3) incorporates eQTLs identified in different tissues; and (4) uses simulations to account for multiple testing. We applied this approach to 2 published asthma GWASs (combined n = 46,044) and used mouse studies to provide initial functional insights into 2 genes with novel genetic associations., Results: We tested the association between asthma and 17,190 genes that were found to have cis- and/or trans-eQTLs across 16 published eQTL studies. At an empirical FDR of 5%, 48 genes were associated with asthma risk. Of these, for 37, the association was driven by eQTLs located in established risk loci for allergic disease, including 6 genes not previously implicated in disease cause (eg, LIMS1, TINF2, and SAFB). The remaining 11 significant genes represent potential novel genetic associations with asthma. The association with 4 of these replicated in an independent GWAS: B4GALT3, USMG5, P2RY13, and P2RY14, which are genes involved in nucleotide synthesis or nucleotide-dependent cell activation. In mouse studies, P2ry13 and P2ry14-purinergic receptors activated by adenosine 5-diphosphate and UDP-sugars, respectively-were upregulated after allergen challenge, notably in airway epithelial cells, eosinophils, and neutrophils. Intranasal exposure with receptor agonists induced the release of IL-33 and subsequent eosinophil infiltration into the lungs., Conclusion: We identified novel associations between asthma and eQTLs for 4 genes related to nucleotide synthesis/signaling and demonstrated the power of gene-based analyses of GWASs., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The cytokine receptor-like factor 2 (CRLF2) gene plays an important role in early B-cell development. Aberrations in CRLF2 activate the JAK-STAT signaling pathway that contributes to B-cell acute lymphoblastic leukemia (B-ALL). The prognostic significance of CRLF2 overexpression and P2RY8-CRLF2 fusion in various B-ALL risk subgroups has not been well established. Two hundred seventy-one patients with newly diagnosed childhood B-ALL were enrolled from a Chinese population. The prevalence of CRLF2 overexpression, CRLF2-P2RY8 fusion, CRLF2 F232C mutation, and JAK2 and IL7R mutational status were analyzed, and the prognostic impact of CRLF2 overexpression and P2RY8-CRLF2 on B-ALL was evaluated by assessing their influence on overall survival and event-free survival. CRLF2 overexpression and P2RY8-CRLF2 were found in 19% and 10%, respectively, in the whole cohort. No correlation between CRLF2 overexpression and P2RY8-CRLF2 was observed. CRLF2 F322C and IL7R mutations were not detected in B-ALL cases overexpressing CRLF2, and no JAK2 mutations were found in the whole cohort either. The results showed that CRLF2 overexpression and P2RY8-CRLF2 were associated with a poor outcome in unselected B-ALL. Moreover, in an intermediate risk B-ALL subgroup P2RY8-CRLF2 was correlated with worse survival, whereas in high- and low-risk subgroups, CRLF2 overexpression predicted a poor outcome. Our findings suggest that P2RY8-CRLF2 is an independent prognostic indicator in intermediate risk B-ALL, while CRLF2 overexpression is correlated with an inferior outcome in high- or low-risk B-ALL. Our study demonstrates that the impact of P2RY8-CRLF2 and CRLF2 overexpression on B-ALL survival may differ across risk subgroups. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background/aims: Patients with acute coronary syndromes (ACS) presenting with cardiogenic shock (CS) are at particular risk for death and adverse cardiac events. Impaired effects and absorption of oral P2Y12-receptor inhibitors due to decreased organ hypoperfusion or hypothermia and challenges regarding oral administration contribute to this risk. We report a single center experience regarding the use of intravenous P2Y12-receptor inhibitor cangrelor in patients with CS treated with percutaneous coronary intervention (PCI)., Methods: Twelve patients with ACS and CS undergoing PCI, not pretreated with oral P2Y12-receptor inhibitors, were treated with cangrelor. Platelet inhibition was assessed by multiple electrode aggregometry (MEA) before and after PCI, immediately and 2 hours after stopping the cangrelor infusion., Results: Nine patients recovered from their cardiogenic shock, 3 patients died. Platelet reactivity decreased from 65.9 (SD 41.0) U before PCI to 15.8 (SD 10.8) U after PCI, 13.4 (SD 7.7) U at the end of infusion and 33.8 (SD 19.9) 2 hours after stopping the cangrelor infusion. There was no non-responder under cangrelor infusion (MEA < 46 U)., Conclusions: Due to its favorable PK/PD profile, cangrelor overcomes problems with reduced absorption and effects of oral P2Y12-receptor inhibitors and should be considered for periprocedural treatment of patients with CS., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Background/aims: Extracellular ATP performs multiple important functions via activation of P2 receptors on the cell surface. P2Y receptors play critical roles in ATP evoked response in human lung adenocarcinoma cells (A549 cells). Emodin is an anthraquinone derivative originally isolated from Chinese rhubarb, possesses anticancer properties. In this study we examined the inhibiting effects of emodin on proliferation, migration and epithelial-mesenchymal transition (EMT) by suppressing P2Y receptors-dependent Ca2+ increase and nuclear factor-κB (NF-KB) signaling in A549 cells., Methods: A549 cells were pretreated with emodin before stimulation with ATP for the indicated time. Then, intracellular Ca2+ concentration ([Ca2+]i) was measured by Fluo-8/AM staining. Cell proliferation and cell cycle progression were tested by CCK8 assay and flow cytometry In addition, wound healing and western blot were performed to determine cell migration and related protein levels (Bcl-2, Bax, claudin-1, NF-κB)., Results: Emodin blunted ATP/UTP-induced increase of [Ca2+]i and cell proliferation concentration-dependently Meanwhile, it decreased ATP-induced cells accumulation in the S phase. Furthermore, emodin altered protein abundance of Bcl-2, Bax and claudin-1 and attenuated EMT caused by ATP. Such ATP-induced cellular reactions were also inhibited by a nonselective P2Y receptors antagonist, suramin, in a similar way to emodin. Besides, emodin could inhibit activation of NF-κB, thus suppressed ATP-induced proliferation, migration and EMT., Conclusion: Our results demonstrated that emodin inhibits ATP-induced proliferation, migration, EMT by suppressing P2Y receptors-mediated [Ca2+]i increase and NF-κB signaling in A549 cells., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) are expressed in virtually all cells with implications in very diverse biological functions, including the well-established platelet aggregation (P2Y12), but also immune regulation and inflammation. The classical P2Y receptors bind nucleotides and are encoded by eight genes with limited sequence homology, while phylogenetically related receptors (e.g., P2Y12-like) recognize lipids and peptides, but also nucleotide derivatives. Growing lines of evidence suggest an important function of P2Y receptors in immune cell differentiation and maturation, migration, and cell apoptosis. Here, we give a perspective on the P2Y receptors' molecular structure and physiological importance in immune cells, as well as the related diseases and P2Y-targeting therapies. Extensive research is being undertaken to find modulators of P2Y receptors and uncover their physiological roles. We anticipate the medical applications of P2Y modulators and their immune relevance., (© 2017 Elsevier Inc. All rights reserved.)

Cutaneous wound healing is accelerated by mechanical stretching, and treatment with hyperforin, a major component of a traditional herbal medicine and a known TRPC6 activator, further enhances the acceleration. We recently revealed that this was due to the enhancement of ATP-Ca 2+ signaling in keratinocytes by hyperforin treatment. However, the low aqueous solubility and easy photodegradation impede the topical application of hyperforin for therapeutic purposes. We designed a compound hydroxypropyl- β -cyclodextrin- (HP- β -CD-) tetracapped hyperforin, which had increased aqueous solubility and improved photoprotection. We assessed the physiological effects of hyperforin/HP- β -CD on wound healing in HaCaT keratinocytes using live imaging to observe the ATP release and the intracellular Ca 2+ increase. In response to stretching (20%), ATP was released only from the foremost cells at the wound edge; it then diffused to the cells behind the wound edge and activated the P2Y receptors, which caused propagating Ca 2+ waves via TRPC6. This process might facilitate wound closure, because the Ca 2+ response and wound healing were inhibited in parallel by various inhibitors of ATP-Ca 2+ signaling. We also applied hyperforin/HP- β -CD on an ex vivo skin model of atopic dermatitis and found that hyperforin/HP- β -CD treatment for 24 h improved the stretch-induced Ca 2+ responses and oscillations which failed in atopic skin., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse.

Arterial blood pressure is controlled by vasodilatory factors such as nitric oxide (NO) that are released from the endothelium under the influence of fluid shear stress exerted by flowing blood. Flow-induced endothelial release of ATP and subsequent activation of Gq/G11-coupled purinergic P2Y2 receptors have been shown to mediate fluid shear stress-induced stimulation of NO formation. However, the mechanism by which fluid shear stress initiates these processes is unclear. Here, we have shown that the endothelial mechanosensitive cation channel PIEZO1 is required for flow-induced ATP release and subsequent P2Y2/Gq/G11-mediated activation of downstream signaling that results in phosphorylation and activation of AKT and endothelial NOS. We also demonstrated that PIEZO1-dependent ATP release is mediated in part by pannexin channels. The PIEZO1 activator Yoda1 mimicked the effect of fluid shear stress on endothelial cells and induced vasorelaxation in a PIEZO1-dependent manner. Furthermore, mice with induced endothelium-specific PIEZO1 deficiency lost the ability to induce NO formation and vasodilation in response to flow and consequently developed hypertension. Together, our data demonstrate that PIEZO1 is required for the regulation of NO formation, vascular tone, and blood pressure., Competing Interests: The authors have declared that no conflict of interest exists.

Uridine adenosine tetraphosphate (Up4A), a dinucleotide, exerts vascular influence via purinergic receptors (PR). We investigated the effects of Up4A on angiogenesis and the putative PR involved. Tubule formation assay was performed in a three-dimensional system, in which human endothelial cells were cocultured with pericytes with various Up4A concentrations for 5 days. Expression of PR subtypes and angiogenic factors was assessed in human endothelial cells with and without P2Y6R antagonist. No difference in initial tubule formation was detected between Up4A stimulation and control conditions at day 2 In contrast, a significant increase in vascular density in response to Up4A was observed at day 5 Up4A at an optimal concentration of 5 μM promoted total tubule length, number of tubules, and number of junctions, all of which were inhibited by the P2Y6R antagonist MRS2578. Higher concentrations of Up4A (10 μM) had no effects on angiogenesis parameters. Up4A increased mRNA level of P2YRs (P2Y2R, P2Y4R, and P2Y6R) but not P2XR (P2X4R and P2X7R) or P1R (A2AR and A2BR), while Up4A upregulated VEGFA and ANGPT1, but not VEGFR2, ANGPT2, Tie1, and Tie2. In addition, Up4A increased VEGFA protein levels. Transcriptional upregulation of P2YRs by Up4A was inhibited by MRS2578. In conclusion, Up4A is functionally capable of promoting tubule formation in an in vitro coculture system, which is likely mediated by pyrimidine-favored P2YRs but not P2XRs or P1Rs, and involves upregulation of angiogenic factors., (Copyright © 2016 the American Physiological Society.)

Ethnopharmacological Relevance: Fructus gardenia is widely used for treatment of stroke and infectious diseases in Chinese medicine. Geniposide is the key bioactive compound related to the pharmacodynamic actions of gardenia on ischemic stroke. The molecular mechanism by which geniposide improves the ischemic brain injury was observed in the study., Aim of the Study: Recent studies showed that geniposide had protective activities against the inflammatory response in ischemic stroke. However, the molecular mechanism of geniposide anti-inflammatory role has not yet been fully elucidated. In this study, we investigated the effect of geniposide on the expression of P2Y14 receptor and downstream signaling pathway in brain microvascular endothelial cells (BMECs)., Materials and Methods: An in vitro model of cerebral ischemia in BMECs was established by oxygen-glucose-deprivation (OGD). To further confirm the specific effect of geniposide on P2Y14 receptor and downstream signaling pathways, we set up a UDP-glucose (an agonist of the P2Y14 receptor) stimulated model. After administration of geniposide, the expression of P2Y14 receptor, phosphorylation of RAF-1, mitogen activated protein kinase kinase1/2 (MEK1/2), extracellular signal-regulated kinase 1/2 (ERK1/2), level of interleukin-8 (IL-8), interleukin-1β (IL-1β), monocyte chemotactic protein 1 (MCP-1) in BMECs were determined., Results: The mRNA and protein expression of P2Y14 in the rat BMECs were up-regulated in OGD-induced injury. After administration of Geniposide, the expression of P2Y14 receptor was significantly down-regulated, the phosphorylation of RAF-1, MEK1/2, ERK1/2 were suppressed. Similar data were obtained in UDP-glc stimulated model. We also observed that geniposide markedly declined the production of IL-8, IL-1β and MCP-1 in OGD-induced BMECs., Conclusion: Geniposide exerted anti-inflammatory effects by interfering with the expression of P2Y14 receptor, which subsequently inhibits the downstream ERK1/2 signaling pathways and the release of the pro-inflammatory cytokines IL-8, MCP-1, IL-1β. Therefore, this study provides the evidence for gardenia's clinical application in cerebral ischemia., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Hearing and its protection is regulated by ATP-evoked Ca(2+) signaling in the supporting cells of the organ of Corti, however, the unique anatomy of the cochlea hampers observing these mechanisms. For the first time, we have performed functional ratiometric Ca(2+) imaging (fura-2) in three different supporting cell types in the hemicochlea preparation of hearing mice to measure purinergic receptor-mediated Ca(2+) signaling in pillar, Deiters' and Hensen's cells. Their resting [Ca(2+)]i was determined and compared in the same type of preparation. ATP evoked reversible, repeatable and dose-dependent Ca(2+) transients in all three cell types, showing desensitization. Inhibiting the Ca(2+) signaling of the ionotropic P2X (omission of extracellular Ca(2+)) and metabotropic P2Y purinergic receptors (depletion of intracellular Ca(2+) stores) revealed the involvement of both receptor types. Detection of P2X2,3,4,6,7 and P2Y1,2,6,12,14 receptor mRNAs by RT-PCR supported this finding and antagonism by PPADS suggested different functional purinergic receptor population in pillar versus Deiters' and Hensen's cells. The sum of the extra- and intracellular Ca(2+)-dependent components of the response was about equal with the control ATP response (linear additivity) in pillar cells, and showed supralinearity in Deiters' and Hensen's cells. Calcium-induced calcium release might explain this synergistic interaction. The more pronounced Ca(2+) leak from the endoplasmic reticulum in Deiters' and Hensen's cells, unmasked by cyclopiazonic acid, may also suggests the higher activity of the internal stores in Ca(2+) signaling in these cells. Differences in Ca(2+) homeostasis and ATP-induced Ca(2+) signaling might reflect the distinct roles these cells play in cochlear function and pathophysiology.

Background: A deregulated CRLF2 (d-CRLF2) expression was described in B-cell acute lymphoblastic leukemia without recurrent fusion genes (B-NEG ALL). While the role of d-CRLF2 in children has been extensively described, little is known about its role and impact in adult ALL., Methods: Expression levels of CRLF2 were evaluated by quantitative real-time PCR in 102 newly-diagnosed adult B-NEG ALL and correlated with the clinico-biological characteristics and outcome. Incidence and clinical impact of the P2RY8/CRLF2 transcript was also assessed., Results: High CRLF2 levels, as continuous variable, were significantly associated with hyperleucocytosis (p=0.0002) and thrombocytopenia (p=0.005); when a cut-point at ΔCt≤8 was applied, 35 cases (34.3%), mostly males (80%), proved positive for CRLF2 expression. High CRLF2 levels, as continuous or categorical variable, were associated with a worse disease-free (p=0.003 and p=0.015) and overall survival (p=0.017 and 0.0038). Furthermore, when CRLF2 was analyzed as a categorical variable, a high statistical association was found with IKZF1 deletion and mutations in the JAK/STAT pathway (p=0.001 and p<0.0001, respectively). Finally, the P2RY8/CRLF2 transcript, identified in 8/102 patients (7.8%), was associated with a poor outcome., Conclusions: In adult B-NEG ALL, high CRLF2 expression is associated with distinct clinico-biological features and an unfavourable prognosis in both univariate and multivariate analysis; similarly, P2RY8/CRLF2 positivity correlates with a poor outcome. The quantification of CRLF2 is an important prognostic marker in adult B-lineage ALL without known genetic lesions., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Adenosine 5'-triphosphate (ATP) can act as an extracellular signal that regulates various cellular functions. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in intracellular Ca(2+) ([Ca(2+)]i) and amylase secretion in mouse parotid glands. ATP induced a steep increase in [Ca(2+)]i in acinar cells. The removal of extracellular Ca(2+) or the use of Ca(2+) channel blockers slightly inhibited this increase. Inhibition of PLCγ by U73122 and of IP3 by xestospongin C did not completely block this increase. The purinoceptor antagonists suramin and reactive blue-2 strongly inhibited the ATP-induced changes in [Ca(2+)]i. 2-MeSATP induced a strong increase in [Ca(2+)]i, while Bz-ATP induced a small [Ca(2+)]i increase, and UTP and α,β-MeATP had no effect. The potency order of ATP analogs (2-MeSATP > ATP >> UTP) suggested that P2Y1 and P2Y12 play a significant role in the cellular response to ATP. RT-PCR revealed that P2X2,4,7 and P2Y1,2,10,12,14 were expressed in acinar cells. Ca(2+)-dependent exocytotic secretion of amylase was detected in parotid glands. These findings indicated that ATP activates P2Y receptors more than P2X receptors at low concentrations. Thus, P2Y receptors were found to be the main receptors involved in Ca(2+)-related cell homeostasis and amylase secretion in mouse parotid glands.

Roles of ionotropic purinergic (P2X) receptors in chronic pain have been intensively investigated. However, the contribution of metabotropic purinergic (P2Y) receptors to pathological pain is controversial. In the present study, using single cell RT-PCR (reverse transcription-polymerase chain reaction) and single cell nested-PCR techniques, we examined the expression of P2X(2), P2X(3), P2Y(1) and P2Y(2) mRNA transcripts in retrogradely labeled cutaneous sensory neurons from mouse lumber dorsal root ganglia (DRGs) following peripheral inflammation. The percentage of cutaneous sensory neurons expressing P2Y(2) mRNA transcripts increased after complete Freund's adjuvant (CFA) treatment. Particularly, the P2Y(2) mRNA transcripts were more frequently detected in small-diameter cutaneous neurons from CFA-treated mice than those from control mice. Coexpression of P2Y(2) and P2X (P2X(2) or P2X(3)) mRNAs was more frequently observed in cutaneous sensory neurons from CFA-treated mice relative to controls. Pain behavioral tests showed that the blockade of P2Y receptors by suramin attenuated mechanical allodynia evoked either by CFA or uridine triphosphate (UTP), an endogenous P2Y(2) and P2Y(4) agonist. These results suggest that chronic inflammatory pain enhances expression of P2Y(2) receptor in peripheral sensory neurons that innervate the injured tissue and the activation of P2Y receptors contributes to mechanical allodynia following inflammation.

Extracellular adenosine 5'-triphosphate (ATP) is a signaling molecule that induces a plethora of effects ranging from the regulation of cell proliferation to modulation of cancerous cell behavior. In colorectal cancer, ATP was reported to stimulate epithelial cell proliferation and possibly promote resistance to anti-cancer treatments. However, the exact role of this danger-signaling molecule on cancerous intestinal epithelial cells (IECs) in response to chemotherapeutic agents remains unknown. To address how ATP may influence the response of cancerous IECs to chemotherapeutic agents, we used Caco-2 cells, which display enterocyte-like features, to determine the effect of ATP on the expression of multidrug resistance-associated protein 2 (MRP2). Gene and protein expression were determined by quantitative real-time PCR (qRT-PCR) and Western blotting. Resistance to etoposide, cisplatin and doxorubicin was determined by MTT assays in response to ATP stimulation of Caco-2 cells and in cells for which MRP2 expression was down-regulated by shRNA. ATP increased the expression of MRP2 at both the mRNA and protein levels. MRP2 expression involved an ATP-dependent stimulation of the MEK/ERK signaling pathway that was associated with an increase in relative resistance of Caco-2 cells to etoposide. Abolition of MRP2 expression using shRNA significantly reduced the protective effect of MRP2 toward etoposide as well as to cisplatin and doxorubicin. This study describes the mechanism by which ATP may contribute to the chemoresistance of cancerous IECs in colorectal cancer. Given the heterogeneity of colorectal adenocarcinoma responses to anti-cancer drugs, these findings call for further study to understand the role of P2 receptors in cancer drug therapy and to develop novel therapies aimed at regulating P2 receptor activity.

We conducted a large-scale functional genetic study to characterize mechanisms of resistance to ALK inhibition in ALK-dependent lung cancer cells. We identify members of known resistance pathways and additional putative resistance drivers. Among the latter were members of the P2Y purinergic receptor family of G-protein-coupled receptors (P2Y1, P2Y2, and P2Y6). P2Y receptors mediated resistance in part through a protein-kinase-C (PKC)-dependent mechanism. Moreover, PKC activation alone was sufficient to confer resistance to ALK inhibitors, whereas combined ALK and PKC inhibition restored sensitivity. We observed enrichment of gene signatures associated with several resistance drivers (including P2Y receptors) in crizotinib-resistant ALK-rearranged lung tumors compared to treatment-naive controls, supporting a role for these identified mechanisms in clinical ALK inhibitor resistance., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Purpose: Here, we have studied the effects of the dinucleotide P(1), P(4)-Di (adenosine-5') tetraphosphate (Ap4 A) on corneal barrier function conferred by the tight junction (TJ) proteins and its possible involvement in ocular drug delivery and therapeutic efficiency., Experimental Approach: Experiments in vitro were performed using human corneal epithelial cells (HCLEs) treated with Ap4 A (100 μM) for 5 min. Western blot analysis and transepithelial electrical resistance (TEER) were performed to study the TJ protein levels and barrier function respectively. Intracellular pathways involved were determined using an ERK inhibitor and P2Y(2) receptor siRNAs. In in vivo assays with New Zealand rabbits, TJ integrity was examined by zonula occludens-1 (ZO-1) staining. The hypotensive compound 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) was used to assess improved delivery, measuring its levels by HPLC and measuring intraocular pressure using 5-MCA-NAT, P2Y receptor antagonists and P2Y2 siRNAs., Key Results: Two hours after Ap4 A pretreatment, TJ protein levels in HCLE cells were reduced around 40% compared with control. TEER values were significantly reduced at 2 and 4 h (68 and 52% respectively). TJ reduction and ERK activation were blocked by the ERK inhibitor U012 and P2Y(2) siRNAs. In vivo, topical application of Ap4 A disrupted ZO-1 membrane distribution. 5-MCA-NAT levels in the aqueous humour were higher when Ap4 A was previously instilled and its hypotensive effect was also increased. This action was reversed by P2Y receptor antagonists and P2Y(2) siRNA., Conclusions and Implications: Ap4 A increased corneal epithelial barrier permeability. Its application could improve ocular drug delivery and consequently therapeutic efficiency., (© 2014 The British Pharmacological Society.)

We examined ATP-induced intracellular Ca(2+) ([Ca(2+)]i) responses in the neurons and satellite cells from one of the viscerosensory ganglia, the nodose ganglion (NG), as well as the GABA-mediated modulation of ATP-induced neuronal [Ca(2+)]i responses using intracellular calcium imaging. In neurons with satellite cells, ATP induced [Ca(2+)]i increases in both the neurons and satellite cells. The P2X receptor agonist, α,β-meATP, induced [Ca(2+)]i increases in neurons and this response was inhibited by the P2X receptor antagonist, PPADS. On the other hand, the P2Y receptor agonist, ADP, induced [Ca(2+)]i increases in satellite cells, and this response was inhibited by the P2Y receptor antagonist, MRS2179. RT-PCR detected the expression of P2X2, P2X3, P2Y1, and P2Y2 receptor mRNAs in NG extracts. Immunohistochemistry revealed that NG neurons and satellite cells were immunoreactive to P2X2 and P2X3, and P2Y1 and P2Y2 receptors, respectively. In isolated neurons, the ATP-evoked [Ca(2+)]i increase was inhibited by GABA. However, in neurons with satellite cells, the GABAA receptor antagonist, bicuculline, enhanced the ATP-induced [Ca(2+)]i increase in neurons. These results suggest that viscerosensory neuronal excitability may be modulated by GABA from satellite cells in NG., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Alzheimer's disease (AD) is the most common cause of dementia, affecting more than 10% of people over the age of 65. Age is the greatest risk factor for AD, although a combination of genetic, lifestyle and environmental factors also contribute to disease development. Common features of AD are the formation of plaques composed of beta-amyloid peptides (Aβ) and neuronal death in brain regions involved in learning and memory. Although Aβ is neurotoxic, the primary mechanisms by which Aβ affects AD development remain uncertain and controversial. Mouse models overexpressing amyloid precursor protein and Aβ have revealed that Aβ has potent effects on neuroinflammation and cerebral blood flow that contribute to AD progression. Therefore, it is important to consider how endogenous signalling in the brain responds to Aβ and contributes to AD pathology. In recent years, Aβ has been shown to affect ATP release from brain and blood cells and alter the expression of G protein-coupled P2Y receptors that respond to ATP and other nucleotides. Accumulating evidence reveals a prominent role for P2Y receptors in AD pathology, including Aβ production and elimination, neuroinflammation, neuronal function and cerebral blood flow., (© 2014 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Diabetes leads to impairment of the normal course of wound healing. Interestingly, recent studies have implicated a critical role of P2X/P2Y nucleotide receptors in dermal tissue regeneration and maintaining vascular homeostasis. As new vessel generation and keratinization process are decreased in diabetic patients we determined whether nucleoside 5'-O-phosphorothioate analogues might accelerate vascular endothelial growth factor (VEGF) production as well as the growth and migration of human keratinocytes under hyperglycaemic conditions. We also investigated the expression pattern of P2X/P2Y receptors in human keratinocyte HaCaT cells. We show here that nucleoside 5'-Ophosphorothioate analogues are better candidates to overcome hyperglycaemia-induced impairment of angiogenesis as compared to their unmodified counterparts. The greatest potency for VEGF release and stimulation of cell migration by thiophosphate analogues of ATP and UTP correlates with the highest P2Y2 receptor expression by HaCaT cells. We also found that UTPαS significantly increased the viability and proliferation of the HaCaT cells. These findings suggest that thiophosphate analogues of nucleotides could serve as potential therapeutic agents for promoting impaired angiogenesis under diabetic conditions.

Purinergic signalling plays a crucial role in proper functioning of the nervous system. Mechanisms depending on extracellular nucleotides and their P2 receptors also underlie a number of nervous system dysfunctions. This review aims to present the role of purinergic signalling, with particular focus devoted to role of P2 family receptors, in epilepsy, depression, neuropathic pain, nervous system neoplasms, such as glioma and neuroblastoma, neurodegenerative diseases like Parkinson's disease, Alzheimer's disease and multiple sclerosis. The above-mentioned conditions are associated with changes in expression of extracellular ectonucleotidases, P2X and P2Y receptors in neurons and glial cells, as well as releasing considerable amounts of nucleotides from activated or damaged nervous tissue cells into the extracellular space, which contributes to disturbance in purinergic signalling. The numerous studies indicate a potential possibility of using synthetic agonists/antagonists of P2 receptors in treatment of selected nervous system diseases. This is of particular significance, since numerous available agents reveal a low effectiveness and often produce side effects.