Cooperation of Adenosine with Macrophage Toll-4 Receptor Agonists Leads to Increased Glycolytic Flux through the Enhanced Expression of PFKFB3 Gene

Almudena Ruiz-García,Eva Monsalve,Laura Novellasdemunt,Àurea Navarro-Sabaté,Anna Manzano,Samuel Rivero,Antonio Castrillo,Marta Casado,Jorge Laborda,Ramón Bartrons,María José M Díaz-Guerra

doi:10.1074/jbc.m110.190298

Abstract

Macrophages activated through Toll receptor triggering increase the expression of the A(2A) and A(2B) adenosine receptors. In this study, we show that adenosine receptor activation enhances LPS-induced pfkfb3 expression, resulting in an increase of the key glycolytic allosteric regulator fructose 2,6-bisphosphate and the glycolytic flux. Using shRNA and differential expression of A(2A) and A(2B) receptors, we demonstrate that the A(2A) receptor mediates, in part, the induction of pfkfb3 by LPS, whereas the A(2B) receptor, with lower adenosine affinity, cooperates when high adenosine levels are present. pfkfb3 promoter sequence deletion analysis, site-directed mutagenesis, and inhibition by shRNAs demonstrated that HIF1α is a key transcription factor driving pfkfb3 expression following macrophage activation by LPS, whereas synergic induction of pfkfb3 expression observed with the A(2) receptor agonists seems to depend on Sp1 activity. Furthermore, levels of phospho-AMP kinase also increase, arguing for increased PFKFB3 activity by phosphorylation in long term LPS-activated macrophages. Taken together, our results show that, in macrophages, endogenously generated adenosine cooperates with bacterial components to increase PFKFB3 isozyme activity, resulting in greater fructose 2,6-bisphosphate accumulation. This process enhances the glycolytic flux and favors ATP generation helping to develop and maintain the long term defensive and reparative functions of the macrophages.

Highlights

Macrophages are key cellular components of the innate immune system
We show here that LPS-dependent induction of HIF1␣ is essential for pfkfb3 expression, the synergic induction of pfkfb3 expression observed with A2R agonists critically depends on the transcription factor Sp1
Adenosine Cooperates with LPS to Increase pfkfb3 Gene Expression—As macrophage activation by pathogens increases ATP consumption [34], and high metabolic activity leads to the liberation of adenosine [35], we explored whether adenosine could affect pfkfb3 expression and modulate the glycolytic flux in activated macrophages

Summary

The abbreviations used are

Fru-2,6-P2, fructose 2,6-bisphosphate; C/EBP␤, CCAAT/enhancer-binding protein ␤; NECA, 5Ј-(N-ethylcarboxamido)adenosine; A2R, A2 receptor; HRE, hypoxia-response element; CREB, cAMPresponse element-binding protein. Pfkfb mRNA contains multiple copies of the AUUUA instability motif in its 3Ј-nontranslated region [14] This sequence motif confers both enhanced translation and instability to the mRNA molecule, and it plays an important role in regulating the half-life of the gene product in different physiological conditions. Utilization of ATP during periods of high metabolic activity leads to an increased concentration of intracellular adenosine that can be secreted through nucleoside transporters Another major pathway contributing to high extracellular adenosine concentration during metabolic stress is the release from the cells of its precursor adenine nucleotides (ATP, ADP, and AMP), followed by extracellular degradation to adenosine. We show that Toll receptor agonists and adenosine, through its A2A and A2B receptors, cooperate to increase glycolytic flux in macrophages by favoring the expression of the PFKFB3 isozyme. We show here that LPS-dependent induction of HIF1␣ is essential for pfkfb expression, the synergic induction of pfkfb expression observed with A2R agonists critically depends on the transcription factor Sp1

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION