The International Journal of Biochemistry & Cell Biology
The NF-κB member p65 controls glutamine metabolism through miR-23a
Graphical abstract
Highlights
► Glutamine medium induces cell cycle arrest in leukemic cells. ► Glutamine medium activates p65. ► p65 decreases miR-23a expression. ► p65 favors leukemic cell growth in glutamine medium.
Introduction
During tumorigenesis, cells show important alterations in their metabolism that can facilitate tumor progression (Villalba et al., 2012). Cancer cells often elevate aerobic glycolysis, in a process termed the Warburg effect (Warburg, 1956). In addition, a significant proportion of tumor cells also increase glutamine metabolism (DeBerardinis et al., 2007, Dang, 2009, Smolkova et al., 2011). Glutamine is the amino acid with the highest circulating levels in human blood and serves as an important source of cellular energy through mitochondrial oxidative phosphorylation (OXPHOS) and of anabolic carbon and nitrogen (Curthoys and Watford, 1995). Activation of oncogenes or loss of tumor suppressors could drive these tumor metabolic changes. One example is induction of mitochondrial glutaminase (GLS) expression by the oncogene c-Myc (Gao et al., 2009, Wise et al., 2008). The catabolism of glutamine is initiated by either of two isoforms, kidney type and liver type, of GLS (Curthoys and Watford, 1995). Kidney type GLS is abundant in kidney, brain, intestine, fetal liver, lymphocytes and transformed cells. GLS catabolyzes the conversion of glutamine to glutamate, which can enter into the Krebs cycle as α-ketoglutarate (Curthoys and Watford, 1995). Interestingly, GLS is probably the rate-limiting enzyme for glutamine consumption in proliferating T cells as well as leukemic cells (Carr et al., 2010).
By increasing GLS activity, tumor cells can use glutamine not only as an amino acid for protein synthesis or as a nitrogen donor for nucleic acid synthesis, but also as a source of the anti-oxidant glutathione or as an energy source through α-ketoglutarate. It has been known for a long time that lymphocytes and thymocytes consume glutamine at high rates, even higher than glucose rate (Yaqoob and Calder, 1997, Carr et al., 2010). Proliferating T cells show an increase in aerobic glycolysis and glutamine consumption, mainly as an energy source (Brand et al., 1986). This is phenotyped by tumor leukemic cells, which show even higher glutamine uptake and GLS activity than activated primary T cells (Carr et al., 2010).
The mechanisms controlling GLS in tumor cells are poorly understood but it has recently emerged that microRNAs (miRNAs), a class of short, non-coding RNA molecules, regulate GLS-mediated glutamine metabolism. miRNAs play a central role in regulating posttranscriptional gene expression by annealing to the 3′ untranslated regions of target mRNAs to generally promote mRNA degradation or translational repression (Chhabra et al., 2010). c-Myc transcriptionally represses miR-23a and miR-23b, which target GLS mRNA, resulting in greater expression of GLS protein (Gao et al., 2009).
The promoter region of the miR-23a∼27a∼24-2 cluster spans from −603 to +36 bp, it lacks both the known common as well as less common promoter elements i.e. TATA box, the downstream promoter element (DPE), the downstream core element (DCE), and the multiple start site element downstream (MED-1; Chhabra et al., 2010). This is relatively normal since more than 80% of mammalian protein coding genes are driven by TATA less promoter. The miR-23a∼27a∼24-2 promoter is also different from the promoters of RNA Pol II transcribed snRNA genes as it lacks the proximal sequence element (PSE; Chhabra et al., 2010). All these suggest that regulation of this locus could be unconventional. In addition, not all members of miR-23a∼27a∼24-2 locus are equally regulated (Lee et al., 2008, Buck et al., 2010, Chhabra et al., 2010). Transcriptional and post-transcriptional mechanisms could be responsible for these observations, including complex regulatory mechanisms, which can act even after the primary transcript of miR-23a∼27a∼24-2 is produced.
The transcription factor NF-κB plays essential roles in survival and proliferation of primary and leukemic cells (Karin and Greten, 2005, Espinosa et al., 2010, Zhao, 2010). The NF-κB member p65 binds and activates the miR-23b promoter in human biliary epithelial cells (Zhou et al., 2010). However, this study did not investigate GLS expression, which is a bona fide target of miR-23 (Gao et al., 2009). Moreover, p65 induces GLS activity by an unknown mechanism in NIH 3T3 cells (Wang et al., 2010) and recent data suggest that NF-κB links LPS-induced inflammation to mitochondrial biogenesis, where glutamine is primarily metabolized (Suliman et al., 2010). All these data suggest that p65 could positively modulate glutamine metabolism in leukemic cells.
We have recently observed that the MAPK ERK5 is essential for leukemic cell survival in glutamine medium (Charni et al., 2010). Glutamine increases ERK5 expression and activation (Charni et al., 2010). ERK5 activation induces p65 translocation to the nucleus and increases its transcriptional activity (Garaude et al., 2006). Here, we show that cells growing in glutamine increase p65 translocation to the nucleus where it controls glutamine metabolism by downregulating miR-23a levels. This leads to an increased GLS expression. Taken together, all these results suggest that p65 manages leukemic cell metabolism. Constitutive activation of NF-κB in leukemic cells could provide them with a selective metabolic advantage.
Section snippets
Reagent and antibodies
Mitotracker Red was from Molecular Probes. Tetramethylrhodamine ethyl ester (TMRE) and leupeptin were from Fluka. The protease inhibitors aprotenin and 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) were from Euromedex. H2O2, glucose oxidase (GO), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), mannitol and sucrose were from SIGMA. Trizol reagent was from Invitrogen. Galactose and glutamine were from GIBCO. The antibodies against Cox IV and β-actin were from Cell Signaling Technology.
The NF-κB p65 member decreased miR-23 expression by recruiting HDAC to miR-23a promoter
In silico analysis of transcription factors that bind to the miR-23a∼27a∼24-2 cluster's promoter showed that it possessed several binding sites for NF-κB members, including p65 (tgGGGAActtcctc, −2008:−2021) and c-Rel (GGAAAtccca, −2010:−2019; GGAAAtccca, −1402:−1411) (Alexiou et al., 2009). Hence, we investigated if NF-κB could control glutamine metabolism through miR-23 expression. Overexpression of p65 decreased the activity of a reporter plasmid driven by the 2.4 kb upstream of the miR-23a
Discussion
It is well known that NF-κB is an important regulator of cell survival, proliferation and differentiation and is frequently involved in malignant transformation (Karin, 2006), including in T leukemias (Espinosa et al., 2010, Zhao, 2010). Leukemic cells often show higher glutamine consumption than non-transformed T cells (Carr et al., 2010). Here we show that leukemic cells with high NF-κB p65 subunit activity increase GLS expression and have the metabolic advantage to easily use glutamine. A
Conflict of interest
The authors declare no competing financial or other interests.
Acknowledgements
This work was supported by the program “Chercheur d’avenir” from the Region Languedoc-Rousillon (MV), a scientific program from the “Communauté de Travail des Pyrénées” (CTPP10/09 to MV), the Association pour la Recherche contre le Cancer (MV), the Fondation pour la Recherche Medicale (MV), a grant FEDER Objectif competitivite (MV), a grant from European Community Program SUDOE (MV) and a fellowships from the ARC and Higher Education Commission, Pakistan (MGR).
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