Synnestvedt K, Furuta GT, Comerford KM, Louis N, Karhausen J, Eltzschig HK et al.. Ecto-5′-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. J Clin Invest 110: 993-1002

Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Journal of Clinical Investigation (Impact Factor: 13.22). 11/2002; 110(7):993-1002. DOI: 10.1172/JCI15337
Source: PubMed


Under conditions of limited oxygen availability (hypoxia), multiple cell types release adenine nucleotides in the form of ATP, ADP, and AMP. Extracellular AMP is metabolized to adenosine by surface-expressed ecto-5'-nucleotidase (CD73) and subsequently activates surface adenosine receptors regulating endothelial and epithelial barrier function. Therefore, we hypothesized that hypoxia transcriptionally regulates CD73 expression. Microarray RNA analysis revealed an increase in CD73 and ecto-apyrase CD39 in hypoxic epithelial cells. Metabolic studies of CD39/CD73 function in intact epithelia revealed that hypoxia enhances CD39/CD73 function as much as 6 +/- 0.5-fold over normoxia. Examination of the CD73 gene promoter identified at least one binding site for hypoxia-inducible factor-1 (HIF-1) and inhibition of HIF-1alpha expression by antisense oligonucleotides resulted in significant inhibition of hypoxia-inducible CD73 expression. Studies using luciferase reporter constructs revealed a significant increase in activity in cells subjected to hypoxia, which was lost in truncated constructs lacking the HIF-1 site. Mutagenesis of the HIF-1alpha binding site resulted in a nearly complete loss of hypoxia-inducibility. In vivo studies in a murine hypoxia model revealed that hypoxia-induced CD73 may serve to protect the epithelial barrier, since the CD73 inhibitor alpha,beta-methylene ADP promotes increased intestinal permeability. These results identify an HIF-1-dependent regulatory pathway for CD73 and indicate the likelihood that CD39/CD73 protects the epithelial barrier during hypoxia.

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    • "It has been described that ectonucleotidases expression is regulated by several transcription factors, including Sp1 [44], Stat3 and Gfi-1 [31]. CD39 and CD73 expression and function are upregulated under hypoxic conditions [45] as well by the presence of TGF-b1 and IL-6 [31] [46], type 1 IFNs [47] and Wnt signaling pathway agonists [48]. In contrast, inflammatory cytokines, such as IL-4, IL-12, IL-21 and IFN-c, can prevent CD73 expression induced by TGF-b1 [46] "
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    ABSTRACT: Extracellular ATP is a danger signal released by dying and damaged cells, and it functions as an immunostimulatory signal that promotes inflammation. However, extracellular adenosine acts as an immunoregulatory signal that modulates the function of several cellular components of the adaptive and innate immune response. Consequently, the balance between ATP and adenosine concentration is crucial in immune homeostasis. CD39 and CD73 are two ectonucleotidases that cooperate in the generation of extracellular adenosine through ATP hydrolysis, thus tilting the balance towards immunosuppressive microenvironments. Extracellular adenosine can prevent activation, proliferation, cytokine production and cytotoxicity in T cells through the stimulation of the A2A receptor; however, recent evidence has shown that adenosine may also affect other processes in T-cell biology. In this review, we discuss evidence that supports a role of CD73 and CD39 ectonucleotidases in controlling naive T-cell homeostasis and memory cell survival through adenosine production. Finally, we propose a novel hypothesis of a possible role of these ectonucleotidases and autocrine adenosine signaling in controlling T-cell differentiation. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Jul 2015 · FEBS Letters
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    • "Although the endpoints of such signaling vary, significant emphasis has been placed on the barrier-protective aspects of hypoxia and HIF signaling (Colgan and Taylor, 2010; Glover and Colgan, 2011). To date, HIF target genes implicated in such barrier protection are more " nonclassical " in nature, including intestinal trefoil factor (Furuta et al., 2001), multidrug resistance gene (Comerford et al., 2002), mucin-3 (Louis et al., 2006), and CD73 (Synnestvedt et al., 2002). "
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    ABSTRACT: Intestinal epithelial cells (IECs) are exposed to profound fluctuations in oxygen tension and have evolved adaptive transcriptional responses to a low-oxygen environment. These adaptations are mediated primarily through the hypoxia-inducible factor (HIF) complex. Given the central role of the IEC in barrier function, we sought to determine whether HIF influenced epithelial tight junction (TJ) structure and function. Initial studies revealed that short hairpin RNA-mediated depletion of the HIF1β in T84 cells resulted in profound defects in barrier and nonuniform, undulating TJ morphology. Global HIF1α chromatin immunoprecipitation (ChIP) analysis identified claudin-1 (CLDN1) as a prominent HIF target gene. Analysis of HIF1β-deficient IEC revealed significantly reduced levels of CLDN1. Overexpression of CLDN1 in HIF1β-deficient cells resulted in resolution of morphological abnormalities and restoration of barrier function. ChIP and site-directed mutagenesis revealed prominent hypoxia response elements in the CLDN1 promoter region. Subsequent in vivo analysis revealed the importance of HIF-mediated CLDN1 expression during experimental colitis. These results identify a critical link between HIF and specific tight junction function, providing important insight into mechanisms of HIF-regulated epithelial homeostasis.
    Full-text · Article · Jun 2015 · Molecular Biology of the Cell
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    • "These discrepancies suggest that the microenvironment and maturation state of T cells may be critical for the expression of purinergic receptors and the ectonucleotide cascade. In line with this interpretation, maturation and activation state of T cells was reported to influence ectoenzyme expression levels [26], [27]. "
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    ABSTRACT: Extracellular nucleotides and nucleosides have been implicated as important signaling molecules in the pathogenesis of acute lung injury (ALI). While adenosine is known to inhibit T cell activation, little information is available as to ATP and NAD degrading enzymes, the expression of ATP and adenosine receptors/transporters in different T cell subsets. ALI was induced by challenging mice with intra-tracheal instillation of 60 µl (3 µg/g) LPS. After 3 d and 7 d blood, lung tissue and bronchoalveolar lavage was collected and immune cells were analyzed using flow cytometry. The transcriptional phenotype of T helper cells, cytotoxic and regulatory T cells sorted by FACS was assessed by measuring the expression profile of 28 genes related to purinergic signaling using TaqMan Array Micro Fluidic Cards. Catabolism of ATP, NAD and cAMP by activated CD4+ T cells was evaluated by HPLC. CD73 was found to be highly abundant on lymphoid cells with little abundance on myeloid cells, while the opposite was true for CD39. After ALI, the abundance of CD39 and CD73 significantly increased on all T cell subsets derived from lung tissue and bronchoalveolar space. Expression analysis in T cell subsets of the lung revealed ATP (Cd39, Cd73) and NAD (Cd38, Cd157, Cd296, Pc-1) degrading enzymes. However, only transcription of Cd38, Cd39, Cd73, Ent1 and A2a receptor was significantly upregulated after ALI in T helper cells. CD4+ T cells from injured lung rapidly metabolized extracellular ATP to AMP and adenosine but not NAD or cAMP. These findings show that lung T cells - the dominant cell fraction in the later phase of ALI - exhibit a unique expression pattern of purinergic signaling molecules. Adenosine is formed by T cells at an enhanced rate from ATP but not from NAD and together with upregulated A2a receptor is likely to modulate the healing process after acute lung injury.
    Full-text · Article · Apr 2014 · PLoS ONE
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