Anales RANF

S10-04 REGULATION OF P2X7 RECEPTOR GENE EXPRESSION IN NEUROBLASTOMA CELLS R. Gómez-Villafuertes, M. Benito-León, F. Ortega, R. Pérez-Sen, E.G. Delicado, M.T. Miras-Portugal Complutense University of Madrid, Madrid, Spain In the nervous system, P2X7 receptors (P2X7R) are involved not only in physiological functions such as cell growth, differentiation and apoptosis, but also in brain pathologies including neurodegenerative diseases and cancer. P2X7R are highly expressed by nearly all human cancers so far investigated, including neuroblastoma cells. Noticeable, trophic deprivation induces a significant increase in P2rx7 gene expression, facilitating proliferation of neuroblastoma cells in the absence of trophic support. Blockade of P2X7R results in increased neuritogenesis in neuroblastoma cells, whereas P2X7R overexpression significantly reduces the formation of neurites. In resting conditions, P2X7 transcript levels seems to be mainly regulated by the specificity protein 1 (Sp1), which is a multifunctional transcription factor constitutively expressed that directly binds to GC-rich DNA motifs to modify the expression of a wide variety of genes. We have demonstrated that the increase in P2X7R expression following serum withdrawal requires the activation of PI3K/Akt pathway and depends on nuclear Sp1 levels, since blockade of Sp1-dependent transcription with mithramycin A prevents upregulation of P2rx7 gene. Now we have found a new regulatory mechanism of P2rx7 gene expression independent of Sp1, which is mediated by dual specificity phosphatases (DUSPs), enzymes that regulate the activity of mitogen activated kinases (MAPKs). Inhibition of DUPS1/DUSP6 by (E)-2-benzylidene-3- (cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI) strongly increases the expression of P2X7R, effect that is significantly reduced by p38/ERK inhibition. BCI treatment strongly enhances p38 and JNK phosphorylation, but decreases ERK1/2 phosphorylation, suggesting that BCI mainly inhibits DUSP1 activity in neuroblastoma cells. The observed decrease in ERK phosphorylation is mediated by activation of protein phosphatase 2 following p38 phosphorylation. Interestingly, the effect of BCI on P2X7R expression is not only independent of Sp1, but also independent of other transcription factors such as AP-1, CREB and Myc. Work supported by the Spanish Ministry of Economy (BFU 2014-53654-P) and Ramón Areces Fundation (PR2018/16-02). References : Gómez-Villafuertes et al., 2015, Sci Rep 5:18417. García-Huerta et al., 2012, J Biol Chem 287(53):44628-44. Gutiérrez-Martín et al., 2011, J Biol Chem 286(13):11370-81. Gómez-Villafuertes et al., 2009, FEBS J 276(18):5307-25.