Ectonucleoside Triphosphate Diphosphohydrolase Research Articles

MIG-23 is involved in sperm migration by modulating extracellular ATP levels in Ascaris suum.

In nematodes, spermiogenesis is a process of sperm activation in which nonmotile spermatids are transformed into crawling spermatozoa. Sperm motility acquisition during this process is essential for successful fertilization, but the underlying mechanisms remain to be clarified. Herein, we have found that extracellular adenosine-5'-triphosphate (ATP) level regulation by MIG-23, which is a homolog of human ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), was required for major sperm protein (MSP) filament dynamics and sperm motility in the nematode Ascaris suum. During sperm activation, a large amount of ATP was produced in mitochondria and was stored in refringent granules (RGs). Some of the produced ATP was released to the extracellular space through innexin channels. MIG-23 was localized in the sperm plasma membrane and contributed to the ecto-ATPase activity of spermatozoa. Blocking MIG-23 activity resulted in a decrease in the ATP hydrolysis activity of spermatozoa and an increase in the depolymerization rate of MSP filaments in pseudopodia, which eventually affected sperm migration. Overall, our data suggest that MIG-23, which contributes to the ecto-ATPase activity of spermatozoa, regulates sperm migration by modulating extracellular ATP levels.

ATP and Adenosine Metabolism in Cancer: Exploitation for Therapeutic Gain.

Adenosine is an evolutionary ancient metabolic regulator linking energy state to physiologic processes, including immunomodulation and cell proliferation. Tumors create an adenosine-rich immunosuppressive microenvironment through the increased release of ATP from dying and stressed cells and its ectoenzymatic conversion into adenosine. Therefore, the adenosine pathway becomes an important therapeutic target to improve the effectiveness of immune therapies. Prior research has focused largely on the two major ectonucleotidases, ectonucleoside triphosphate diphosphohydrolase 1/cluster of differentiation (CD)39 and ecto-5'-nucleotidase/CD73, which catalyze the breakdown of extracellular ATP into adenosine, and on the subsequent activation of different subtypes of adenosine receptors with mixed findings of antitumor and protumor effects. New findings, needed for more effective therapeutic approaches, require consideration of redundant pathways controlling intratumoral adenosine levels, including the alternative NAD-inactivating pathway through the CD38-ectonucleotide pyrophosphatase phosphodiesterase (ENPP)1-CD73 axis, the counteracting ATP-regenerating ectoenzymatic pathway, and cellular adenosine uptake and its phosphorylation by adenosine kinase. This review provides a holistic view of extracellular and intracellular adenosine metabolism as an integrated complex network and summarizes recent data on the underlying mechanisms through which adenosine and its precursors ATP and ADP control cancer immunosurveillance, tumor angiogenesis, lymphangiogenesis, cancer-associated thrombosis, blood flow, and tumor perfusion. Special attention is given to differences and commonalities in the purinome of different cancers, heterogeneity of the tumor microenvironment, subcellular compartmentalization of the adenosine system, and novel roles of purine-converting enzymes as targets for cancer therapy. SIGNIFICANCE STATEMENT: The discovery of the role of adenosine as immune checkpoint regulator in cancer has led to the development of novel therapeutic strategies targeting extracellular adenosine metabolism and signaling in multiple clinical trials and preclinical models. Here we identify major gaps in knowledge that need to be filled to improve the therapeutic gain from agents targeting key components of the adenosine metabolic network and, on this basis, provide a holistic view of the cancer purinome as a complex and integrated network.

Abstract 321: Inhibition of CD39 results in elevated ATP and activation of myeloid cells to promote anti-tumor immunity

Abstract CD39 (ENTPD1) is an ecto-nucleoside triphosphate diphosphohydrolase expressed widely in the tumor microenvironment (TME) responsible for catalyzing the conversion of ATP to AMP. Inhibition of CD39 enzymatic activity can promote anti-tumor immune responses by increasing the immunostimulatory substrate ATP and decreasing the formation of the product AMP, a precursor to immunoinhibitory adenosine. CD39 inhibition has been shown to have an anti-tumor effect by activating dendritic cells, macrophages, and NK cells in the TME to promote antigen presentation and inflammatory cytokine release. AB598 is a novel antibody, highly specific for human and cynomolgus CD39. AB598 potently binds to CD39 expressed on human primary myeloid cells and tumor cells and potently inhibits both soluble and membrane-bound CD39 enzymatic activity. CD39 inhibition results in increased extracellular ATP (eATP) in the TME and subsequent activation of P2X and P2Y receptors. Myeloid cells highly express both CD39 and P2X7, the P2X receptor with the weakest ligand affinity for ATP (greater than 100 μM), and most likely to be specifically activated by very high levels of eATP resulting from CD39 inhibition. Thus, the myeloid compartment has emerged as a key target associated with the therapeutic effects of CD39 inhibition. AB598 retains the ability to bind and inhibit membrane bound CD39 in the presence of high ATP (400 μM). AB598 promotes maturation of human dendritic cells in the presence of ATP, as determined by increased expression of CD83 and CD86 and decreased expression of CD14. In human macrophages treated with ATP, AB598 activates the inflammasome, resulting in the secretion of pro-inflammatory IL-18 and IL-1β. The ATP-dependent effects of AB598 support a therapeutic rationale of combining it with immunogenic therapies that induce release of ATP, such as chemotherapy and radiation, which might represent a new paradigm in the treatment of advanced solid tumors. Citation Format: Christine E. Bowman, Ada Chen, Damie Juat, Kaustubh Parashar, Julie Clor, Hema Singh, Ritu Kushwaha, Bryan Handlos, Suan Liu, Janine Kline, Xiaoning Zhao, Hyock Joo Kwon, David Green, Stephen W. Young, Ester Fernandez-Salas, Matthew J. Walters, Nigel P. Walker. Inhibition of CD39 results in elevated ATP and activation of myeloid cells to promote anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 321.

Pharmacological Aspects of Natural Quercetin in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that can lead to severe joint damage, disability and mortality. Quercetin (QUE) is a natural flavonoid that is ubiquitous in fruits and vegetables. This article reviews the effect of QUE on articular and extra-articular manifestations of RA in vitro and in vivo. In general, for articular manifestations, QUE inhibited synovial membrane inflammation by reducing inflammatory cytokines and mediators, decreasing oxidative stress, inhibiting proliferation, migration and invasion, and promoting apoptosis of fibroblast-like synoviocytes (FLS), regulated autoimmune response through modulating Th17/Treg imbalance and Th17 cells differentiation, reducing autoantibodies levels and regulating ectonucleoside triphosphate diphosphohydrolase (E-NTPDase)/ectoadenosine deaminase (E-ADA) activities, reduced bony damage via lowering matrix metalloproteinase (MMP)-1, MMP-3, receptor activator of nuclear factor kappa B ligand (RANKL) expression and osteoclasts formation. For extra-articular manifestations, QUE could reverse the neurodegenerative processes of the enteric nervous system (ENS) and exhibited cytoprotective, genoprotective and hepatoprotective effects. In addition, we also summarize some contradictory experimental results and explore the possibility for these differences to form a sound basis for the clinical application of QUE for RA.

Caffeic acid regulates glucose homeostasis and inhibits purinergic and cholinergic activities while abating oxidative stress and dyslipidaemia in fructose-streptozotocin-induced diabetic rats.

The antidiabetic potential of caffeic acid in fructose/streptozotocin-induced type 2 diabetic rats was examined in this study. Male Sprague-Dawley rats were supplied with 10% fructose solution for 14 days followed by an intraperitoneal injection of 40 mg/kg bw streptozotocin to induce type 2 diabetes (T2D). Rats were treated with both low (150 mg/kg bw) and high (300 mg/kg bw) doses of caffeic acid for 5 weeks, while the positive control group was treated with metformin (200 mg/kg bw). Treatment with caffeic acid significantly decreased blood glucose levels and elevated serum insulin levels while improving glucose tolerance, pancreatic β-cell function and morphology. It also led to a significant reduction of serum cholesterol, triglyceride, LDL-cholesterol, ALT, AST, creatinine, urea and uric acid levels, while increasing HDL cholesterol levels. Caffeic acid significantly (P < 0.05) elevated hepatic glycogen level, serum and pancreatic glutathione level, superoxide dismutase and catalase activities with a concomitant decrease in malondialdehyde level, α-amylase, lipase, adenosine triphosphatase (ATPase), ectonucleoside triphosphate diphosphohydrolase (ENTPDase), 5'-nucleotidase (5'-NTD) and acetylcholinesterase activities. The results suggest caffeic acid as a potent natural product with therapeutic effects against T2D. Further molecular and clinical studies are, however, required to ascertain these findings.

Generation, breakdown and responses of ATP in the carotid body

Purinergic signaling involving adenosine triphosphate (ATP) acting on P2X2/3 receptors modulates physiological carotid body (CB) afferent discharge and chemoreflex activation however, the upregulation of P2X3 receptors on petrosal sensory neurons is partly responsible for aberrant CB tonicity and hyperreflexia, and the increase in blood pressure in the Spontaneously Hypertensive rat (SHR). The enhanced chemoreflex occurs prior to the onset of hypertension and elevated CB activity is thought to contribute to the underlying pathophysiology of the disease but the exact mechanisms remain unclear. We hypothesize that there is greater release of ATP and/or decreased ATP breakdown in the pre‐hypertensive SHR CB compared with that in the Wistar rat, and this evokes an increase in the chemoreflex response and subsequent activation of the sympathetic nervous system. We performed three studies. (i) Using digital droplet PCR (ddPCR), we measured mRNA expression levels of enzymes involved in the breakdown of ATP in CBs extracted from male 4‐week‐old prehypertensive SHR and Wistar rats; these included: ectonucleotide pyrophosphatase/phosphodiesterase 1‐3 (Enpp1‐3), ectonucleoside triphosphate diphosphohydrolase 2‐3 (Entpd2‐3), and ecto‐5’‐nucleotidase (Nt5e). (ii) We quantified ATP release from CBs in vitro during baseline conditions and during cyanide‐evoked ATP release using a colorimetric assay (213‐579‐1 ‐ Millipore), comparing between male 4‐week‐old pre‐hypertensive SHR and Wistar rats. (iii) In an in situ working heart‐brainstem preparation of 4‐week‐old Wistar rats, simultaneous recordings were made from phrenic (PN), hypoglossal (HN), recurrent laryngeal (RLN), abdominal (ABN), and thoracic sympathetic nerves (tSNA). The peripheral chemoreceptors were stimulated with either potassium cyanide (KCN 20‐100 μL, 0.04%) or ATP at varying concentrations (10‐100 µL, 50 µM ‐ 2 mM) delivered via the internal carotid artery. In whole CB extracts, ddPCR revealed increased Enpp2‐3 and Nt5e expression (p&lt;0.05) in pre‐hypertensive SHRs relative to Wistar rats suggesting greater capacity to break down ATP in the pre‐hypertensive SHR CB. There were no differences in the expressions of Enpp1 or Entpd2‐3 between rat strains. However, the amount of ATP released from the CB in both baseline and during cyanide stimulation was two‐fold greater in the pre‐hypertensive SHR (P&lt;0.05). Stimulation of the CB with KCN evoked hyperpnoea, bradycardia and sympathoexcitation at all doses. In contrast, CB injection of low dose ATP increased PN rate with little effect on tSNA, whereas higher doses induced apnea accompanied by repetitive burst discharge of post‐inspiratory activity in the RLN with tonic elevations in the discharge of both ABN and tSNA. In sum, despite the upregulation of several ATP degrading enzymes in the CBs of the SHR, CB release of ATP is higher in this species. We hypothesize that the increase in ATP release in the SHR CB drives the CB hyperexcitability and that the shift towards an upregulation of ATP degrading enzymes even prior to the development of hypertension is compensatory to oppose the increase in ATP signaling. The dose‐dependent effect of exogenous ATP at supraphysiological levels evoking greater CB‐mediated expiratory and sympathetic outflow in the Wistar rat mimics aspects of pathophysiological CB chemoreflex signaling in the SHR.

Amniotic fluid stem cell-derived extracellular vesicles are independent metabolic units capable of modulating inflammasome activation in THP-1 cells.

An immunoregulatory role of stem cells, often mediated by their secretome, has been claimed by several studies. Stem cell-derived extracellular vesicles (EVs) are crucial components of the secretome. EVs, a heterogeneous group of membranous vesicles released by many cell types into the extracellular space, are now considered as an additional mechanism for intercellular communication. In this study, we aimed at investigating whether human amniotic stem cell-derived extracellular vesicles (HASC-EVs) were able to interfere with inflammasome activation in the THP-1 cell line. Two subsets of HASC-EVs were collected by sequential centrifugation, namely HASC-P10 and HASC-P100. We demonstrated that HASC-EVs were neither internalized into nor undertake a direct interaction with THP-1 cells. We showed that HASC-P10 and P100 were able to intrinsically produce ATP, which was further converted to adenosine by 5'-nucleotidase (CD73) and ectonucleoside triphosphate diphosphohydrolase-1 (CD39). We found that THP-1 cells conditioned with both types of HASC-EVs failed to activate the NLRP3/caspase-1/inflammasome platform in response to LPS and ATP treatment by a mechanism involving A2a adenosine receptor activation. These results support a role for HASC-EVs as independent metabolic units capable of modifying the cellular functions, leading to anti-inflammatory effects in monocytic cells.

CD73 controls ocular adenosine levels and protects retina from light-induced phototoxicity

ATP and adenosine have emerged as important signaling molecules involved in vascular remodeling, retinal functioning and neurovascular coupling in the mammalian eye. However, little is known about the regulatory mechanisms of purinergic signaling in the eye. Here, we used three-dimensional multiplexed imaging, in situ enzyme histochemistry, flow cytometric analysis, and single cell transcriptomics to characterize the whole pattern of purine metabolism in mouse and human eyes. This study identified ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2, and ecto-5′-nucleotidase/CD73 as major ocular ecto-nucleotidases, which are selectively expressed in the photoreceptor layer (CD73), optic nerve head, retinal vasculature and microglia (CD39), as well as in neuronal processes and cornea (CD39, NTPDase2). Specifically, microglial cells can create a spatially arranged network in the retinal parenchyma by extending and retracting their branched CD39high/CD73low processes and forming local “purinergic junctions” with CD39low/CD73− neuronal cell bodies and CD39high/CD73− retinal blood vessels. The relevance of the CD73–adenosine pathway was confirmed by flash electroretinography showing that pharmacological inhibition of adenosine production by injection of highly selective CD73 inhibitor PSB-12489 in the vitreous cavity of dark-adapted mouse eyes rendered the animals hypersensitive to prolonged bright light, manifested as decreased a-wave and b-wave amplitudes. The impaired electrical responses of retinal cells in PSB-12489-treated mice were not accompanied by decrease in total thickness of the retina or death of photoreceptors and retinal ganglion cells. Our study thus defines ocular adenosine metabolism as a complex and spatially integrated network and further characterizes the critical role of CD73 in maintaining the functional activity of retinal cells.

Expression of Ectonucleoside Triphosphate Diphosphohydrolase 2 (NTPDase2) Is Negatively Regulated Under Neuroinflammatory Conditions In Vivo and In Vitro.

Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) hydrolyzes extracellular ATP to ADP, which is the ligand for P2Y1,12,13 receptors. The present study describes the distribution of NTPDase2 in adult rat brains in physiological conditions, and in hippocampal neurodegeneration induced by trimethyltin (TMT). The study also describes the regulation of NTPDase2 by inflammatory mediators in primary astrocytes and oligodendroglial cell line OLN93. In physiological conditions, NTPDase2 protein was most abundant in the hippocampus, where it was found in fibrous astrocytes and synaptic endings in the synaptic-rich hippocampal layers. In TMT-induced neurodegeneration, NTPDase2-mRNA acutely decreased at 2-dpi and then gradually recovered to the control level at 7-dpi and 21-dpi. As determined by immunohistochemistry and double immunofluorescence, the decrease was most pronounced in the dentate gyrus (DG), where NTPDase2 withdrew from the synaptic boutons in the polymorphic layer of DG, whereas the recovery of the expression was most profound in the subgranular layer. Concerning the regulation of NTPDase2 gene expression, proinflammatory cytokines IL-6, IL-1β, TNFα, and IFNγ negatively regulated the expression of NTPDase2 in OLN93 cells, while did not altering the expression in primary astrocytes. Different cell-intrinsic stressors, such as depletion of intracellular energy store, oxidative stress, endoplasmic reticulum stress, and activation of protein kinase C, also massively disturbed the expression of the NTPDase2 gene. Together, our results suggest that the expression and the activity of NTPDase2 transiently cease in neurodegeneration and brain injury, most likely as a part of the acute adaptive response designed to promote cell defense, survival, and recovery.

Differential regulation of E-NTPdases during Leishmania amazonensis lifecycle and effect of their overexpression on parasite infectivity and virulence.

Infections caused by Leishmania amazonensis are characterized by a persistent parasitemia due to the ability of the parasite to modulate the immune response of macrophages. It has been proposed that ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDases) could be able to suppress the host immune defense by reducing the ATP and ADP levels. The AMP generated from E-NTPDase activity can be subsequently hydrolyzed by ecto-nucleotidases, increasing the levels of adenosine, which can reduce the inflammatory response. In the present work, we provide new information about the role of E-NTPDases on infectivity and virulence of L. amazonensis. Our data demonstrate that not only the E-NTPDase activity is differentially regulated during the parasite development but also the expression of the genes ntpd1 and ntpd2. E-NTPDase activity increases significantly in axenic amastigotes and metacyclic promastigotes, both infective forms in mammalian host. A similar profile was found for mRNA levels of the ntpd1 and ntpd2 genes. Using parasites overexpressing the genes ntpd1 and ntpd2, we could demonstrate that L. amazonensis promastigotes overexpressing ntpd2 gene show a remarkable increase in their ability to interact with macrophages compared to controls. In addition, both ntpd1 and ntpd2-overexpressing parasites were more infective to macrophages than controls. The kinetics of lesion formation by transfected parasites were similar to controls until the second week. However, twenty days post-infection, mice infected with ntpd1 and ntpd2-overexpressing parasites presented significantly reduced lesions compared to controls. Interestingly, parasite load reached similar levels among the different experimental groups. Thus, our data show a non-linear relationship between higher E-NTPDase activity and lesion formation. Previous studies have correlated increased ecto-NTPDase activity with virulence and infectivity of Leishmania parasites. Based in our results, we are suggesting that the induced overexpression of E-NTPDases in L. amazonensis could increase extracellular adenosine levels, interfering with the balance of the immune response to promote the pathogen clearance and maintain the host protection.

Discovery proteomics defines androgen-regulated glycoprotein networks in prostate cancer cells, as well as putative biomarkers of prostatic diseases

Supraphysiologic androgen (SPA) inhibits cell proliferation in prostate cancer (PCa) cells by transcriptional repression of DNA replication and cell-cycle genes. In this study, quantitative glycoprotein profiling identified androgen-regulated glycoprotein networks associated with SPA-mediated inhibition of PCa cell proliferation, and androgen-regulated glycoproteins in clinical prostate tissues. SPA-regulated glycoprotein networks were enriched for translation factors and ribosomal proteins, proteins that are known to be O-GlcNAcylated in response to various cellular stresses. Thus, androgen-regulated glycoproteins are likely to be targeted for O-GlcNAcylation. Comparative analysis of glycosylated proteins in PCa cells and clinical prostate tissue identified androgen-regulated glycoproteins that are differentially expressed prostate tissues at various stages of cancer. Notably, the enzyme ectonucleoside triphosphate diphosphohydrolase 5 was found to be an androgen-regulated glycoprotein in PCa cells, with higher expression in cancerous versus non-cancerous prostate tissue. Our glycoproteomics study provides an experimental framework for characterizing androgen-regulated proteins and glycoprotein networks, toward better understanding how this subproteome leads to physiologic and supraphysiologic proliferation responses in PCa cells, and their potential use as druggable biomarkers of dysregulated AR-dependent signaling in PCa cells.

Fc Receptor-Dependent Trogocytosis of CD39 Impacts Engraftment and Invasiveness of Acute Myeloid Leukemia Cells

Corresponding author: Dr. Simon. C. Robson (srobson@bidmc.harvard.edu).Introduction: CD39/ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1) is the prototypic member of the GDA1-CD39 superfamily of ectonucleotidases and modulates purinergic signaling pathways. CD39 expression has been noted in human acute myeloid leukemia (AML) and likely contributes to chemoresistance [1]. Our study reported here elucidates the impact of Cd39 on engraftment and invasiveness of AML TIB-49 cells using an immunocompetent murine experimental model.Methods: Wild-type (WT) mice and Cd39 -/- mice on C57BL/6 background were bred at Beth Israel Deaconess Medical Center. The syngeneic murine AML cell line TIB-49 (Cd39 negative in vitro) was purchased from American Type Culture Collection. For bioluminescence imaging experiments, TIB-49 cells were transduced with luciferase/mCherry using a lentiviral vector. For AML model, mice were administered with 1×10 6 TIB-49-luciferase cells intravenously via tail vein injection. For chloroma model, mice were subcutaneously inoculated with 1×10 6 TIB-49 cells in the right flank. Bioluminescence imaging of TIB-49-luciferase bearing mice was conducted with the IVIS TM 50 Imaging System. Blood, spleen and bone marrow (BM) were also collected from TIB-49 bearing AML mice for FACS (fluorescence activated cell sorting) analysis. To explore Cd39 in TIB engraftment and invasiveness, TIB-49 cells were further transduced with a lentiviral vector overexpressing mCd39 with TdTomato. WT mice were intravenously inoculated with 1×10 6 of either TIB-49-TdTomato cells or TIB-49-mCd39-TdTomato cells, and the above read-outs were determined. To investigate the potential of CD39 as a therapeutic target, we engineered anti-mouse Cd39 antibodies (αCd39 mAb) with isotype selection and removal of fucose to further promote Fc receptor (FcR) interactions.Results: Bioluminescence imaging results indicated that TIB-49 engraftment was decreased in global Cd39 -/- mice with decreased disease burdens noted relative to WT (Figure 1A). FACS analysis of blood, spleen and BM-derived cells from TIB-49 bearing AML-model mice (day 31) confirmed higher engraftment of TIB-49 cells (TdTomato+) at all sites in WT compared to Cd39 -/- mice (Figure 1B). TIB-49 cells did not express Cd39 in vitro, but TIB-49 cells harvested from spleen and BM of WT but not Cd39 -/- mice displayed high levels of Cd39. This indicated TIB-49 cells acquired Cd39 from host cells, in a process of antibody-independent trogocytosis (Figure 1C), as RT-PCR did not detect Cd39 mRNA expression in TIB-49 cells in vivo. Additionally, circulating TIB-49 cells from the blood of WT mice were Cd39 negative (Figure 1C), suggesting a role for the tumor microenvironment in mediating trogocytosis. TIB-49 cells expressing host Cd39 in WT mice spleen and BM lost Cd39 after being exposed to αCd39 mAb treatment. Cd39 translocated from TIB-49 cells to effector cells, at least in part, dependent on FcR mediated trogocytosis (Figure 1D). When Cd39 was overexpressed on TIB-49 cells (TIB-49-mCd39-TdTomato), the engraftment was boosted in WT mice in vivo when compared to TIB-49-TdTomato cells (day 19, Figure 1E) with higher levels of Cd39 expression than that observed on TIB-49-TdTomato cells in spleen and BM (day 26) (Figure 1F). Moreover, TIB-49-mCd39-TdTomato bearing mice displayed shorter survival times, when compared with TIB-49-TdTomato bearing AML mice (Figure 1G). The αCd39 mAb monotherapy had no effect on TIB-49 chloroma model growth. However, pretreatment with αCd39 mAb effectively boosted daunorubicin chemotherapeutic effects in vivo (Figure 1H and 1I).Conclusions: Our study suggests bidirectional trogocytosis between TIB-49 AML and host immune cells, which is further modulated by FcR interaction. Re-distribution of Cd39 from host to TIB-49 cells or induced high level expression contributes to engraftment and invasiveness, resulting in decreased survival. Targeting CD39 is a potential therapeutic approach, operational not only by boosting chemosensitivity but furthering anti-leukemic effects in experimental models.Disclosures: No relevant conflicts of interest to declare.

The Role of GATA3 in Breast Cancer: An Updated Review

GATA binding protein 3 (GATA3), a member of the zinc finger-binding transcription factor GATA gene family, plays an important role in regulating the development and differentiation of various tissues and organs. Previous studies have manifested that GATA3 is implicated in inhibiting the development of breast cancer, including suppressing the growth, metastasis, and invasion of cancer cells. However, the exact role of GATA3 in breast cancer remains to be fully elucidated. Interestingly, recent studies have further shown that GATA3 can regulate a variety of new target genes, including ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3), semaphorin 3B (SEMA3B), and microRNAs (miRNAs) to promote tumorigenesis, epithelial-mesenchymal transformation (EMT), and metastasis of breast cancer. Meanwhile, GATA3 mutations can affect the role of GATA3 in inhibiting the growth and metastasis of breast cancer cells, which indicates that GATA3 might be a promising target for breast cancer treatment. Undoubtedly, these new evidence further uncover the complexity of the GATA3/targets network in breast cancer development. In this article, we review related research progress and put forward some questions about the development of GATA3 in the future in order to be helpful for the understanding on the exact role of GATA3 in the progress of breast cancer and the development of related new clinical treatment strategies that will ultimately benefit the clinical outcome of breast cancer patients.

Morphology and chemical characteristics of taste buds associated with P2X3-immunoreactive afferent nerve endings in the rat incisive papilla.

The present study investigated the cellular components and afferent innervations of taste buds in the rat incisive papilla by immunohistochemistry using confocal scanning laser microscopy. Taste buds containing guanine nucleotide-binding protein G(t), subunit α3 (GNAT3)-imunoreactive cells were densely distributed in the lateral wall of incisive papilla forming the opening of nasoincisor ducts. GNAT3-immunoreactive cells in the taste buds were slender in shape and the tips of apical processes gathered at one point at the surface of the epithelium. The number of taste buds was 56.8±4.5 in the incisive papilla. The incisive taste buds also contained ectonucleoside triphosphate diphosphohydrolase 2-immunoreactive cells and synaptotagmin-1-immunoreactive cells in addition to GNAT3-immunoreactive cells. Furthermore, GNAT3-immunoreactive cells were immunoreactive to taste transduction molecules such as phospholipase C, β2-subunit, and inositol 1,4,5-trisphosphate receptor, type 3. P2X3-immunoreactive subepithelial nerve fibers intruded into the taste buds and terminated with hederiform or calix-like nerve endings attached to GNAT3-immunoreactive cells and synaptosomal-associated protein, 25 kDa-immunoreactive cells. Some P2X3-immunoreactive endings were also weakly immunoreactive for P2X2. Furthermore, a retrograde tracing method using fast blue dye indicated that most of the P2X3-immunoreactive nerve endings originated from the geniculate ganglia (GG) of the facial nerve. These results suggest that incisive taste buds are morphologically and cellularly homologous to lingual taste buds and are innervated by P2X3-immunoreactive nerve endings derived from the GG. The incisive papilla may be the palatal taste papilla that transmits chemosensory information in the oral cavity to the GG via P2X3-immunoreactive afferent nerve endings.

Extracellular ectonucleotidases are differentially regulated in murine tissues and human polymorphonuclear leukocytes during sepsis and inflammation.

Sepsis is life-threatening organ dysfunction caused by a dysregulated inflammatory and immune response to infection. Sepsis involves the combination of exaggerated inflammation and immune suppression. During systemic infection and sepsis, the liver works as a lymphoid organ with key functions in regulating the immune response. Extracellular nucleotides are considered damage-associated molecular patterns and are involved in the control of inflammation. Their levels are finely tuned by the membrane-associated ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) enzyme family. Although previous studies have addressed the role of NTPDase1 (CD39), the role of the other extracellular NTPDases, NTPDase2, -3, and -8, in sepsis is unclear. In the present studies we identified NTPDase8 as a top downregulated gene in the liver of mice submitted to cecal ligation-induced sepsis. Immunohistochemical analysis confirmed the decrease of NTPDase8 expression at the protein level. In vitro mechanistic studies using HepG2 hepatoma cells demonstrated that IL-6 but not TNF, IL-1β, bacteria, or lipopolysaccharide are able to suppress NTPDase8 gene expression. NTPDase8, as well as NTPDase2 and NTPDase3 mRNA was downregulated, whereas NTPDase1 (CD39) mRNA was upregulated in polymorphonuclear leukocytes from both inflamed and septic patients compared to healthy controls. Although the host's inflammatory response of polymicrobial septic NTPDase8 deficient mice was no different from that of wild-type mice, IL-6 levels in NTPDase8 deficient mice were higher than IL-6 levels in wild-type mice with pneumonia. Altogether, the present data indicate that extracellular NTPDases are differentially regulated during sepsis.

The ATP-hydrolyzing ectoenzyme E-NTPD8 attenuates colitis through modulation of P2X4 receptor–dependent metabolism in myeloid cells

Extracellular adenosine triphosphate (ATP) released by mucosal immune cells and by microbiota in the intestinal lumen elicits diverse immune responses that mediate the intestinal homeostasis via P2 purinergic receptors, while overactivation of ATP signaling leads to mucosal immune system disruption, which leads to pathogenesis of intestinal inflammation. In the small intestine, hydrolysis of luminal ATP by ectonucleoside triphosphate diphosphohydrolase (E-NTPD)7 in epithelial cells is essential for control of the number of T helper 17 (Th17) cells. However, the molecular mechanism by which microbiota-derived ATP in the colon is regulated remains poorly understood. Here, we show that E-NTPD8 is highly expressed in large-intestinal epithelial cells and hydrolyzes microbiota-derived luminal ATP. Compared with wild-type mice, Entpd8-/- mice develop more severe dextran sodium sulfate-induced colitis, which can be ameliorated by either the depletion of neutrophils and monocytes by injecting with anti-Gr-1 antibody or the introduction of P2rx4 deficiency into hematopoietic cells. An increased level of luminal ATP in the colon of Entpd8-/- mice promotes glycolysis in neutrophils through P2x4 receptor-dependent Ca2+ influx, which is linked to prolonged survival and elevated reactive oxygen species production in these cells. Thus, E-NTPD8 limits intestinal inflammation by controlling metabolic alteration toward glycolysis via the P2X4 receptor in myeloid cells.

A Recombinant Fusion Construct between Human Serum Albumin and NTPDase CD39 Allows Anti-Inflammatory and Anti-Thrombotic Coating of Medical Devices.

Medical devices directly exposed to blood are commonly used to treat cardiovascular diseases. However, these devices are associated with inflammatory reactions leading to delayed healing, rejection of foreign material or device-associated thrombus formation. We developed a novel recombinant fusion protein as a new biocompatible coating strategy for medical devices with direct blood contact. We genetically fused human serum albumin (HSA) with ectonucleoside triphosphate diphosphohydrolase-1 (CD39), a promising anti-thrombotic and anti-inflammatory drug candidate. The HSA-CD39 fusion protein is highly functional in degrading ATP and ADP, major pro-inflammatory reagents and platelet agonists. Their enzymatic properties result in the generation of AMP, which is further degraded by CD73 to adenosine, an anti-inflammatory and anti-platelet reagent. HSA-CD39 is functional after lyophilisation, coating and storage of coated materials for up to 8 weeks. HSA-CD39 coating shows promising and stable functionality even after sterilisation and does not hinder endothelialisation of primary human endothelial cells. It shows a high level of haemocompatibility and diminished blood cell adhesion when coated on nitinol stents or polyvinylchloride tubes. In conclusion, we developed a new recombinant fusion protein combining HSA and CD39, and demonstrated that it has potential to reduce thrombotic and inflammatory complications often associated with medical devices directly exposed to blood.

Favorable function of Ectonucleoside triphosphate diphosphohydrolase 1 high expression in thyroid carcinoma

BackgroundEctonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) has been proved to play a vital role in human cancers. Nevertheless, the exact role of ENTPD1 in thyroid carcinoma (THCA) remained unclear. This study aimed to evaluate its prognostic value and reveal the potential regulatory mechanism in THCA.Results(1) Higher expression of ENTPD1 was found in THCA tissue compared with normal tissue (all P < 0.05). ENTPD1 expression was associated with age, sub-type and clinical stage of THCA patients (all P < 0.05). Immunohistochemistry showed its higher expression in patients with early stage. (2) ENTPD1 high expression was associated with favorable overall survival of THCA patients (all P < 0.05), especially for male patients and those with advanced stage, B-cells and Natural killer T-cells decreased (all P < 0.05). (3) Pathway analysis indicated that ENTPD1 mainly participated in metabolic process and negatively regulated metabolism-related pathway such as butanoate metabolism, pyruvate metabolism and fatty acid metabolism ((all P < 0.05). (4) ENTPD1 appeared genetic alteration in THCA, and the main mutation type of ENTPD1 was missense substitution (15.89%). (5) A weak correlation between ENTPD1 expression and methylation was found (P < 0.001). Methylation of ENTPD1 in THCA was lower than in normal group (P < 0.001), but it did not correlate with any clinical phenotypes of THCA patients.ConclusionsENTPD1 was highly expressed in THCA, and ENTPD1 high expression contributed to the prognosis of THCA patients. The present study inferred that ENTPD1 might serve as a metabolism-related gene and play a critical role in THCA through regulating metabolic pathways.

Thrombo-Inflammation: A Focus on NTPDase1/CD39.

There is increasing evidence for a link between inflammation and thrombosis. Following tissue injury, vascular endothelium becomes activated, losing its antithrombotic properties whereas inflammatory mediators build up a prothrombotic environment. Platelets are the first elements to be activated following endothelial damage; they participate in physiological haemostasis, but also in inflammatory and thrombotic events occurring in an injured tissue. While physiological haemostasis develops rapidly to prevent excessive blood loss in the endothelium activated by inflammation, hypoxia or by altered blood flow, thrombosis develops slowly. Activated platelets release the content of their granules, including ATP and ADP released from their dense granules. Ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1)/CD39 dephosphorylates ATP to ADP and to AMP, which in turn, is hydrolysed to adenosine by ecto-5′-nucleotidase (CD73). NTPDase1/CD39 has emerged has an important molecule in the vasculature and on platelet surfaces; it limits thrombotic events and contributes to maintain the antithrombotic properties of endothelium. The aim of the present review is to provide an overview of platelets as cellular elements interfacing haemostasis and inflammation, with a particular focus on the emerging role of NTPDase1/CD39 in controlling both processes.

Hesperidin mitigates inflammation and modulates ectoenzymes activity and some cellular processes in complete Freund's adjuvant-induced arthritic rats.

This study was aimed at assessing the anti-arthritic effects of hesperidin on the inflammatory markers in serum/plasma, ectoenzymes activity in platelet, reactive oxygen species (ROS), apoptosis and cell cycle in bone marrow cells of a rat model of arthritis. Fifty-six adult female Wistar rats (245-274 g) were grouped into eight of seven rats each: control rats given normal saline or 40 mg/kg of hesperidin or 80 mg/kg of hesperidin, 0.2 mg/kg of dexamethasone, arthritic rats given normal saline, or 40 mg/kg of hesperidin or 80 mg/kg of hesperidin, and 0.2 mg/kg of dexamethasone. Myeloperoxidase and nitrate plus nitrite levels were evaluated in the plasma and serum, respectively. The ecto-nucleoside triphosphate diphosphohydrolases, ecto-5'-nucleotidase and ecto-adenosine deaminase activities were assessed in platelets. Subsequently, the cells of the bone marrow were obtained, and the assays for ROS, apoptosis and cell cycle were evaluated using flow cytometry. The results showed that hesperidin mitigated inflammation, modulated adenosine nucleotides and nucleoside hydrolysing enzymes and levels, minimized ROS intracellularly, attenuated apoptotic process and activated cell cycle arrest in arthritic rat. This study suggests that hesperidin could be a natural and promising anti-inflammatory compound for the management of arthritis.