La adenosina incrementa la expresión del gen pfkfb3 mediante los receptores de adenosina a2 y potencia el flujo glucolítico y el aporte energético en macrófagos activados

• Autores: Almudena Ruiz García
• Directores de la Tesis: María José Martínez Díaz Guerra (dir. tes.), Jorge Laborda Fernández (codir. tes.)
• Lectura: En la Universidad de Castilla-La Mancha ( España ) en 2012
• Idioma: español
• Tribunal Calificador de la Tesis: Ramón Bartrons Bach (presid.), Mª José Ruiz Hidalgo (secret.), Eva María Galán Moya (voc.), Marta Casado Pinna (voc.), Mairena Martin Lopez (voc.)
• Materias:
  • Química
    • Bioquímica
      • Inmunoquímica
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• Resumen

  • ¿La adenosina incrementa la expresión del gen Pfkfb3 mediante los receptores de adenosina A2 y potencia el flujo glucolítico y el aporte energético en macrófagos activados¿ Macrophages are key cellular elements of the innate immune system. They recognize pathogen¿s components through signals triggered by Toll receptors. The activation of macrophages depends on energy-requiring pathways, mainly glycolysis. Glycolytic flux is primarily controlled by the enzyme PFK-1, whose most powerful allosteric activator is Fru-2,6-P2. In turn, the synthesis and degradation of Fru-2,6-P2 is catalyzed by PFK-2. Different PFK-2 isozymes, generated from four genes (Pfkfb1-4), have been identified in mammalian tissues. Macrophages express the isozyme PFKFB3, a product of the Pfkfb3 gene. This isoform presents the highest kinase-bisphosphatase activity ratio, thus generating the highest levels of Fru-2,6-P2.

    In the last years, adenosine has been shown to modulate the inflammatory response by limiting macrophage activation. Large amounts of adenosine accumulate at sites of inflammation. Adenosine acts at the cell surface through four different adenosine receptors, which are expressed on macrophages and allow these cells to detect stressful conditions and modulate the cellular functions to adapt to their microenvironment.

    Macrophages activated through Toll receptor triggering increase the expression of the A2A and A2B adenosine receptors. In this study, we explored the effects of adenosine on macrophage activity and inflammation. We found that adenosine receptor activation enhances LPS-induced Pfkfb3 expression, resulting in an increase of the key glycolytic allosteric regulator Fru-2,6-P2 and the glycolytic flux. Using shRNA and differential expression of A2A and A2B receptors, we demonstrate that the A2A receptor mediates, in part, the induction of Pfkfb3 by LPS, whereas the A2B 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 HIF-1¿ is a key transcription factor driving Pfkfb3 expression following macrophage activation by LPS, whereas the synergic induction of Pfkfb3 expression observed by treatment with A2 receptor agonists seems to depend on Sp1 activity.

    Furthermore, the levels of pAMPK 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 Fru-2,6-P2 accumulation. This process enhances the glycolytic flux and favors ATP generation helping to develop and maintain the long-term defensive and reparative functions of macrophages.