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

Abstract

The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-gamma (interferon-gamma). 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-gamma was studied using macrophages from STAT1 knockout mice. IFN-gamma triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-gamma is required for DNA synthesis. Interestingly, IFN-gamma 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-gamma 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-gamma 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-gamma in macrophages.

Expression and Functionality of Anti-Human Immunodeficiency Virus and Anticancer Drug Uptake Transporters in Immune Cells

“…Second, PHA stimulation significantly up-regulated hENTs and hCNT2 in PBMCs and CD4 ϩ T-cells. A similar up-regulation had been described for CNT1 and CNT2 upon stimulation of murine bone marrow macrophages with interferon-␥-␣/␤/␥, tumor necrosis factor-␣, or LPS (Soler et al, 2001(Soler et al, , 2003. In contrast, all of these stimuli slightly down-regulated the expression of ENT1 in murine macrophages (Soler et al, 2001), suggesting that the up-regulatory effect of PHA on hENTs might act through a different pathway.…”

“…Transport studies in MDMs were carried out on 24-well plates as described previously (Soler et al, 2003) by incubating semiconfluent cell monolayers for 10 min in an uptake buffer of choline chloride or sodium chloride supplemented with radiolabeled uridine or cytidine or the organic cation model (MPP ϩ ) at a final concentration of 1 M in all cases. The uptake was measured either in the presence or absence of the equilibrative nucleoside inhibitors NBTI (1 M) or dipyridamole (10 M) in nucleoside uptake experiments and the organic cation inhibitor D-22 (20 M) or the high-affinity competitive substrates Rani and ET at 1 mM in organic cation uptake measurements.…”

Nucleoside transporters in chronic lymphocytic leukaemia

“…Whether the lack of correlation between hENT1 mRNA and hENT1 transport activity in CLL is related to the altered STAT1 phosphorylation status is not known, but, in any case, it would be consistent with the finding that ENT1 activity but not mRNA levels depending on STAT1 function in murine bone marrow macrophages. 61 A lack of correlation between hENT1 mRNA levels and drug transport activity has also been described for the hENT1 transporter in MCL cells. 80 For some reason, little CNT-type transport activity is found in CLL and other B-cell lymphomas, although mRNA for hCNT2 and hCNT3 have been detected.…”

“…60 Indeed, phorbol esters downregulate ENT1 activity in B-cell lines, 47 which is interesting because in murine bone marrow macrophages the downregulation of ENT1 activity triggered by IFN-g is STAT1 dependent. 61 Nucleoside transporter expression may also be modulated by nucleosides and nucleoside-derived drugs by other means, probably involving interference with endogenous nucleotide metabolism. Fludarabine and 2-CdA seem to upregulate hENT1 expression in CLL cells, as determined by NBTI binding, probably as a result of an increase in the percentage of cells in S phase.…”

Identification and Characterization of Proximal Promoter Polymorphisms in the Human Concentrative Nucleoside Transporter 2 (SLC28A2)

“…In addition, the expression of the CNT2 in hepatocytes and macrophages has led to an understanding of the endogenous physiological role of CNT2 in the salvage of nucleosides during cell proliferation . The other roles of CNT2 in cell physiology, for example in modulating purinergic responses during cell activation and/or apoptosis, have also been investigated (Soler et al, 2003;Ferná ndez-Veledo et al, 2006). Previously, our group has functionally characterized coding region variants of CNT2 that were identified in an ethnically diverse sample population (Owen et al, 2005).…”