Anales RANF

P.71 INHIBITION OF DUAL-SPECIFICITY PHOSPHATASES PROMOTES P2X7 RECEPTOR EXPRESSION IN NEUROBLASTOMA CELLS IN A p38-DEPENDENT MANNER M. Benito-León, F. Ortega, R. Pérez-Sen, E.G. Delicado, J. Gualix, M.T. Miras- Portugal, R. Gómez-Villafuertes Complutense University of Madrid, Madrid, Spain. The expression of the purinergic receptor P2X7 (P2X7R) in neuroblastoma cells is associated with accelerated growth rate, angiogenesis, metastasis and poor prognosis. Also, an increase in P2X7R expression prolongs the survival of neuroblastoma cells under restrictive conditions, including serum or glucose deprivation. Previously, we identified the specificity protein 1 (Sp1) as the main transcription factor involved in basal expression of P2rx7 gene in N2a neuroblastoma cells. Here, we show a new regulatory mechanism of P2rx7 gene expression mediated by dual specificity phosphatases (DUSPs). DUSPs tightly modulate the activity of mitogen activated kinases (MAPKs) by dephosphorylation of the threonine-X-tyrosine motifs in their activation domains. Treatment of neuroblastoma cells with (E)-2-benzylidene-3- (cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), an allosteric inhibitor of DUSP1 and DUSP6 phosphatases, increases the expression of P2X7R at both transcriptional and protein levels, and this effect is significantly reduced by p38/ERK inhibition. DUSP6 is a constitutive cytosolic phosphatase that mainly dephosphorylates ERK1/2, while DUSP1 belongs to the subgroup of mitogen- or stress-inducible nuclear phosphatases capable of mostly dephosphorylate p38 and JNK. Remarkably, BCI treatment strongly enhances p38 and JNK phosphorylation, while ERK1/2 phosphorylation is reduced, 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. We also demonstrate that the effect of BCI on P2X7R expression is independent of Sp1, as well as other transcription factors such as AP-1, CREB and Myc. Taken together, these results show that neuroblastoma cells have an alternative mechanism independent of Sp1 and dependent on the phosphorylation state of p38/ERK kinases involved in the transcriptional regulation of P2X7R expression. • García-Huerta P, Díaz-Hernandez M, Delicado EG, Pimentel-Santillana M, Miras- Portugal MT, Gómez-Villafuertes R. J Biol Chem. 2012;287(53):44628-44. • Gómez-Villafuertes R, García-Huerta P, Díaz-Hernández JI, Miras-Portugal MT. Sci Rep. 2015;5:18417.