scholarly journals Interferon-γ regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways

2003 ◽  
Vol 375 (3) ◽  
pp. 777-783 ◽  
Author(s):  
Concepció SOLER ◽  
Antonio FELIPE ◽  
José GARCÍA-MANTEIGA ◽  
Maria SERRA ◽  
Elena GUILLÉN-GÓMEZ ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Signal Transduction ◽  
Interferon Γ ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Transcriptional Level ◽  
Nucleoside Transporter ◽  
Protein Levels ◽  
Ifn Γ

The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-γ (interferon-γ). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-γ was studied using macrophages from STAT1 knockout mice. IFN-γ triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-γ is required for DNA synthesis. Interestingly, IFN-γ led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-γ up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-γ is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-γ in macrophages.

IFN-γ + LPS induction of iNOS is modulated by ERK, JNK/SAPK, and p38 mapk in a mouse macrophage cell line

2001 ◽  
Vol 280 (3) ◽  
pp. C441-C450 ◽  
Author(s):  
Edward D. Chan ◽  
David W. H. Riches
Keyword(s):  
Nitric Oxide ◽  
Cell Line ◽  
Inhibitory Effect ◽  
Interferon Γ ◽  
Dominant Negative ◽  
Jnk Pathway ◽  
Mutant Proteins ◽  
Inos Promoter ◽  
Ifn Γ ◽  
Inos Induction

Nitric oxide (NO·) produced by inducible nitric oxide synthase (iNOS) mediates a number of important physiological and pathophysiological processes. The objective of this investigation was to examine the role of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS and NO· by interferon-γ (IFN-γ) + lipopolysaccharide (LPS) in macrophages using specific inhibitors and dominant inhibitory mutant proteins of the MAPK pathways. The signaling pathway utilized by IFN-γ in iNOS induction is well elucidated. To study signaling pathways that are restricted to the LPS-signaling arm, we used a subclone of the parental RAW 264.7 cell line that is unresponsive to IFN-γ alone with respect to iNOS induction. In this RAW 264.7γNO(−) subclone, IFN-γ and LPS are nevertheless required for synergistic activation of the iNOS promoter. We found that extracellular signal-regulated kinase (ERK) augmented and p38 mapk inhibited IFN-γ + LPS induction of iNOS. Dominant-negative MAPK kinase-4 inhibited iNOS promoter activation by IFN-γ + LPS, also implicating the c-Jun NH2-terminal kinase (JNK) pathway in mediating iNOS induction. Inhibition of the ERK pathway markedly reduced IFN-γ + LPS-induced tumor necrosis factor-α protein expression, providing a possible mechanism by which ERK augments iNOS expression. The inhibitory effect of p38 mapk appears more complex and may be due to the ability of p38 mapk to inhibit LPS-induced JNK activation. These results indicate that the MAPKs are important regulators of iNOS-NO· expression by IFN-γ + LPS.

scholarly journals Acquired interferon γ responsiveness during Caco-2 cell differentiation: effects on iNOS gene expression

Gut ◽  
1999 ◽  
Vol 44 (5) ◽  
pp. 659-665 ◽  
Author(s):  
A M Chavez ◽  
M J Morin ◽  
N Unno ◽  
M P Fink ◽  
R A Hodin
Keyword(s):  
Nitric Oxide ◽  
Cell Differentiation ◽  
Interferon Γ ◽  
Intestinal Barrier Function ◽  
Inos Mrna ◽  
Pyrrolidine Dithiocarbamate ◽  
No Production ◽  
Mrna Induction ◽  
Ifn Γ ◽  
Inos Induction

BACKGROUNDImpairment of intestinal barrier function occurs under a variety of inflammatory conditions and is mediated at least in part by interferon γ (IFN-γ) induced nitric oxide (NO) production. Previous in vivo studies have shown that systemic lipopolysaccharide treatment caused an induction of the rat inducible nitric oxide synthase (iNOS) mRNA primarily in villus cells, rather than in undifferentiated crypt cells.AIMSTo examine iNOS induction by IFN-γ in vitro as a function of enterocyte differentiation.METHODSPreconfluent and postconfluent Caco-2 cells were treated with IFN-γ in the presence or absence of various inhibitors. Northern analyses were performed to assess the magnitude of iNOS mRNA induction. IFN-γ receptor mRNA and protein levels were determined.RESULTSiNOS mRNA induction by IFN-γ occurred at two hours and was not blocked by cycloheximide, indicating that it is an immediate early response. iNOS induction and nitrite/nitrate increases were inhibited by dexamethasone and pyrrolidine dithiocarbamate, supporting an important role for the NF-κB transcription factor in this process. The stimulated iNOS induction was seen almost exclusively under conditions of cellular differentiation—that is, in postconfluent Caco-2 cells. This increased IFN-γ responsiveness seen in postconfluent Caco-2 cells correlated with an increased expression of IFN-γ receptor, whereas T84 and HT-29 cells did not show any significant alterations in either iNOS induction or IFN-γ receptor levels as a function of postconfluent growth.CONCLUSIONSWith regard to iNOS mRNA induction, IFN-γ responsiveness is acquired during Caco-2 cell differentiation, perhaps related to an increase in the numbers of IFN-γ receptors.

Interferon-γ switches monocyte differentiation from dendritic cells to macrophages

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 143-150 ◽  
Author(s):  
Yves Delneste ◽  
Peggy Charbonnier ◽  
Nathalie Herbault ◽  
Giovanni Magistrelli ◽  
Gersende Caron ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Interferon Γ ◽  
Transcriptional Level ◽  
Colony Stimulating Factor ◽  
Monocyte Differentiation ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Ifn Γ ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Human monocytes differentiate into dendritic cells (DCs) or macrophages according to the nature of environmental signals. Monocytes stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin 4 (IL-4) yield DCs. We tested here whether interferon-γ (IFN-γ), a potent activator of macrophages, may modulate monocyte differentiation. Addition of IFN-γ to IL-4 plus GM-CSF–stimulated monocytes switches their differentiation from DCs to CD14−CD64+ macrophages. IFN-γ increases macrophage colony-stimulating factor (M-CSF) and IL-6 production by IL-4 plus GM-CSF–stimulated monocytes by acting at the transcriptional level and acts together with IL-4 to up-regulate M-CSF but not IL-6 production. IFN-γ also increases M-CSF receptor internalization. Results from neutralizing experiments show that both M-CSF and IL-6 are involved in the ability of IFN-γ to skew monocyte differentiation from DCs to macrophages. Finally, this effect of IFN-γ is limited to early stages of differentiation. When added to immature DCs, IFN-γ up-regulates IL-6 but not M-CSF production and does not convert them to macrophages, even in the presence of exogenous M-CSF. In conclusion, IFN-γ shifts monocyte differentiation to macrophages rather than DCs through autocrine M-CSF and IL-6 production. These data show that IFN-γ controls the differentiation of antigen-presenting cells and thereby reveals a new mechanism by which IFN-γ orchestrates the outcome of specific immune responses.

scholarly journals Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells

1998 ◽  
Vol 330 (2) ◽  
pp. 997-1001 ◽  
Author(s):  
Antonio FELIPE ◽  
Raquel VALDES ◽  
Belén del SANTO ◽  
Jorge LLOBERAS ◽  
Javier CASADO ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Polyclonal Antibodies ◽  
High Specificity ◽  
Liver Cells ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Nucleoside Transporter ◽  
Transport Activity ◽  
Plasma Membrane Vesicles

Hepatocytes show a Na+-dependent nucleoside transport activity that is kinetically heterogeneous and consistent with the expression of at least two independent concentrative Na+-coupled nucleoside transport systems (Mercader et al. Biochem. J. 317, 835-842, 1996). So far, only a single nucleoside carrier-related cDNA (SPNT) has been isolated from liver cells (Che et al. J. Biol. Chem. 270, 13596-13599, 1995). This cDNA presumably encodes a plasma membrane protein responsible for Na+-dependent purine nucleoside transport activity. Thus, the liver must express, at least, a second nucleoside transporter which should be pyrimidine-preferring. Homology cloning using RT-PCR revealed that a second isoform is indeed present in liver. This second isoform turned out to be identical to the ‘epithelial-specific isoform’ called cNT1, which shows in fact high specificity for pyrimidine nucleosides. Although cNT1 mRNA is present at lower amounts than SPNT mRNA, the amounts of cNT1 protein, when measured using isoform-specific polyclonal antibodies, were even higher than the SPNT protein levels. Moreover, partially purified basolateral plasma membrane vesicles from liver were enriched in the SPNT but not in the cNT1 protein, which suggests that the subcellular localization of these carrier proteins is different. SPNT and cNT1 protein amounts in crude membrane extracts from 6 h-regenerating rat livers are higher than in the preparations from sham-operated controls (3.5- and 2-fold, respectively). These results suggest that liver parenchymal cells express at least two different isoforms of concentrative nucleoside carriers, the cNT1 and SPNT proteins, which show differential regulation and subcellular localization.

scholarly journals Nucleoside transport and proliferative rate in human thymocytes and lymphocytes

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2038-2042 ◽  
Author(s):  
CL Smith ◽  
LM Pilarski ◽  
ML Egerton ◽  
JS Wiley
Keyword(s):  
Cytosine Arabinoside ◽  
Binding Sites ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
S Phase ◽  
Nucleoside Transporters ◽  
Labeling Index ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Equilibrative Nucleoside Transporter

The thymus is a site of active T-lymphoid cell proliferation and DNA synthesis. In this study, the capacity of human thymocytes for nucleoside transport was assessed both by cytosine arabinoside influx and by equilibrium binding of nitrobenzylmercaptopurine riboside (NBMPR), a specific ligand for the equilibrative nucleoside transporter of leukocytes. The proportion of freshly isolated thymocytes synthesizing DNA was 8.6% +/- 2.1% (n = 12) by 3H-thymidine labeling index and 7.8% +/- 2.9% (n = 4) S-phase cells by flow cytometric analysis of DNA content. In comparison, both methods gave proliferation S-phase values less than 1% for peripheral blood lymphocytes (PBLs). Thymocytes expressed a high density of specific NBMPR binding sites (26,068 +/- 8,776 sites per cell, n = 12) as compared with PBLs (1,123 +/- 553 sites per cell, n = 8). The initial influx of cytosine arabinoside into thymocytes was 14-fold greater than into PBLs, and in both cell types the influx of nucleoside was totally inhibited by 0.5 mumol/L NBMPR, which is known to inhibit the major equilibrative nucleoside transporter in white blood cells. Depletion of mature CD3+ cells from the thymocyte preparation by anti-CD3 antibody left a residual population with both increased labeling index and up to twofold greater density of NBMPR binding sites. When PBLs were cultured for 48 hours with the T-cell mitogen phytohemagglutinin, a 40-fold increase in labeling index was observed, together with a 30-fold increase in the density of specific NBMPR binding sites. Thus, fresh thymocytes from human thymus are actively proliferating and express high densities of a functional nucleoside transporter. The more immature cells in the thymocyte population which are proliferating more actively have a greater density of nucleoside transporters than the whole population. In contrast, mitotically inactive PBLs-have few nucleoside transporters, but after mitogenic stimulation PBLs express large numbers of this transmembrane molecule.

In Vitro and In Vivo Evidence That the PP2A Inhibitor SET Regulates IFN-γ Production in Monokine-Stimulated Natural Killer Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 928-928 ◽  
Author(s):  
Rossana Trotta ◽  
Jessica Dal Col ◽  
Jeffrey Allard ◽  
Paolo Neviani ◽  
Ramasamy Santhanam ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Interferon Γ ◽  
Oligonucleotide Array ◽  
Pp2a Activity ◽  
Protein Levels ◽  
Ifn Γ ◽  
Protein Phosphatase Type

Abstract Monokines (i.e. IL-12, IL-18 and IL-15) induce natural killer (NK) cells to produce interferon-γ (IFN-γ), which is critical for monocyte clearance of infectious pathogens and tumor surveillance. To identify new regulators of IFN-γ production we performed oligonucleotide array analysis of unstimulated and IL-12- and IL-18-stimulated NK92 cells. Among the subset of mRNAs differentially regulated in monokine-stimulated cells, we found SET, a potent inhibitor of the protein phosphatase type 2A (PP2A). SET mRNA and/or protein levels were upregulated in IL-12/IL-18- and IL-12/IL-15-stimulated primary human NK cells. Interestingly, the SET protein is also selectively increased in the resting CD56bright NK subset, which is a potent producer of IFN-γ relative to the CD56dim NK subset. To determine whether SET positively regulates IFN-γ production by inhibiting PP2A activity, we employed RNAi and interfered with SET expression in NK92 cells. SET downregulation inhibited IFN-γ secretion by IL-12/IL-18, IL-12/IL-15- or IL-15/IL-18-stimulated NK92 cells. By contrast, ectopic SET expression increased IFN-γ production in monokine-stimulated NK92 and primary human NK cells. Because downregulation of SET augmented PP2A activity in NK92 cells, we sought to investigate whether pharmacologic activation of PP2A inhibits the ability of NK cells to produce IFN-γ. Indeed, suppression of IFN-γ expression and secretion was also observed upon treatment of NK92 and primary NK cells with 1,9-dideoxy-forskolin, a known inducer of PP2A activity. Accordingly, NK cells from mice treated with 1.9-dideoxy-forskolin produced less IFN-γ in response to in vivo monokine stimulation than did NK cells from vehicle-treated mice. Mechanistically, activation of PP2A by SET knock-down or 1,9-dideoxy-forskolin treatment leads to inhibition of ERK1/2, p65RelA and STAT5 activity in monokine-stimulated NK cells. Because these signaling molecules are important for IFN-γ production by monokine-stimulated NK cells, our results strongly suggest that monokine induction of SET expression in NK cells is essential for limiting PP2A activity that, otherwise, would negatively impact the ability of NK cells to produce and release optimal levels of IFN-γ.

Characteristics of membrane transport losses during reticulocyte maturation

1987 ◽  
Vol 65 (10) ◽  
pp. 869-875 ◽  
Author(s):  
Rhoda Blostein ◽  
Eva Grafova
Keyword(s):  
Membrane Transport ◽  
Binding Sites ◽  
Sodium Pump ◽  
Proteolytic Processing ◽  
Atp Depletion ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Nucleoside Transporter ◽  
Transport Losses

The decline in activity of distinct membrane transport systems was followed during in vitro maturation of sheep reticulocytes, namely the sodium pump (measured as specific ouabain binding sites), Na+–glycine cotransport, and the nucleoside transporter (measured as specific nitrobenzylthioinosine binding sites). Certain features of this maturation-associated decline in membrane transport are clarified. Thus, the apparent retardation of loss by metabolic (ATP) depletion, reported previously for the sodium pump and Na+–glycine cotransport, is applicable also to the decline in nucleoside transport. The absolute losses, as well as relative effects of ATP depletion, are different for the three distinct systems. Inhibitors of membrane recycling and (or) intracellular processing, such as chloroquine, as well as ATP depletion, prevent not only the loss but also cause a transient increase in nucleoside transport sites apparent at the surface. Proteolytic processing, at least in the case of the nucleoside transporter, is probably also involved since leupeptin retards the loss in binding sites. Protection against the decline in transporters can also be affected by specific ligands as evidenced in ouabain protection of sodium pump sites. The results provide evidence that membrane transporter recycling is a fundamental process underlying the energy-dependent, maturation-associated loss in membrane transport functions.

Nucleoside transporters and liver cell growth

1998 ◽  
Vol 76 (5) ◽  
pp. 771-777 ◽  
Author(s):  
Marçal Pastor-Anglada ◽  
Antonio Felipe ◽  
F Javier Casado ◽  
Belén Del Santo ◽  
João F Mata ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Growth ◽  
Liver Cell ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Liver Growth ◽  
Liver Parenchymal ◽  
Hepatocyte Regeneration ◽  
Parenchymal Cells

Liver parenchymal cells show a wide variety of plasma membrane transporters that are tightly regulated by endocrine and nutritional factors. This review summarizes work performed in our laboratory on these transport systems, particularly nucleoside transporters, which are up-regulated in physiological situations associated with liver cell growth. Rat hepatocytes show a Na+-dependent nucleoside transport activity that is stimulated by pancreatic hormones. Indeed, this biological activity appears to be the result of the co-expression of at least two isoforms of nucleoside carriers, CNT1 and CNT2 (also called SPNT). These two transporters are up-regulated during the early phase of liver growth after partial hepatectomy, although to different extents, suggesting differential regulation of the two isoforms. The recent generation of isoform-specific antibodies allowed us to demonstrate that carrier expression may also have complex post-transcriptional regulation on the basis of the lack of correspondence between mRNA and protein levels. The analysis of nucleoside transport systems in hepatoma cells and the comparison with those in hepatocytes has also provided evidence that the differentiation status of liver parenchymal cells may determine the pattern of nucleoside transporters expressed.Key words: liver, hepatocyte, regeneration, cell cycle, nucleoside, plasma membrane, transport systems.

scholarly journals Subtype-specific regulation of equilibrative nucleoside transporters by protein kinase CK2

2005 ◽  
Vol 386 (2) ◽  
pp. 281-289 ◽  
Author(s):  
Meaghan STOLK ◽  
Elizabeth COOPER ◽  
Greg VILK ◽  
David W. LITCHFIELD ◽  
James R. HAMMOND
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Interacting Protein ◽  
Human Osteosarcoma Cells ◽  
Specific Regulation ◽  
Membrane Lifetime ◽  
Kinase Ck2

Two subtypes of equilibrative transporters, es (equilibrative inhibitor-sensitive) and ei (equilibrative inhibitor-insensitive), are responsible for the majority of nucleoside flux across mammalian cell membranes. Sequence analyses of the representative genes, ENT1 {equilibrative nucleoside transporter 1; also known as SLC29A1 [solute carrier family 29 (nucleoside transporters), member 1]} and ENT2 (SLC29A2), suggest that protein kinase CK2-mediated phosphorylation may be involved in the regulation of es- and ei-mediated nucleoside transport. We used human osteosarcoma cells transfected with catalytically active or inactive α′ and α subunits of CK2 to assess the effects of CK2 manipulation on nucleoside transport activity. Expression of inactive CK2α′ (decreased CK2α′ activity) increased the number of binding sites (∼1.5-fold) for the es-specific probe [3H]NBMPR ([3H]nitrobenzylthioinosine), and increased (∼1.8-fold) the Vmax for 2-chloro[3H]adenosine of the NBMPR-sensitive (es) nucleoside transporter. There was a concomitant decrease in the Vmax of the NBMPR-resistant (ei-mediated) uptake of 2-chloro[3H]adenosine. This inhibition of CK2α′ activity had no effect, however, on either the KD of [3H]NBMPR binding or the Km of 2-chloro[3H]adenosine uptake. Quantitative PCR showed a transient decrease in the expression of both hENT1 (human ENT1) and hENT2 mRNAs within 4–12 h of induction of the inactive CK2α′ subunit, but both transcripts had returned to control levels by 24 h. These data suggest that inhibition of CK2α′ reduced ei activity by attenuation of hENT2 transcription, while the increase in es/hENT1 activity was mediated by post-translational action of CK2. The observed modification in es activity was probably due to a CK2α′-mediated change in the phosphorylation state of the ENT1 protein, or an interacting protein, effecting an increase in the plasma membrane lifetime of the transport proteins.

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