Cyclic AMP-dependent down regulation of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) in rat C6 glioma

In this communication, we demonstrate that an increase in intracellular cAMP by 1) addition of dibutyrylic cAMP (dbcAMP), a membrane-permeable cAMP-analogue, or 2) activation of the beta-adrenoceptor with (-)-isoproterenol, down regulates the levels of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) mRNA, NPP1 protein and ecto-NPPase activity in rat C6 glioma cells. DbcAMP and (-)-isoproterenol inhibit NPP1 expression in a time and dose-dependent manner. After 48 h of stimulation, 1 mM dbcAMP or 5 mu M (-)-isoproterenol decreases the amount of NPP1 protein by 75 +/- 3% and 81 +/- 1% respectively. Contrary to down regulation of NPP1, we observe an up regulation of glial fibrillary acidic protein (GFAP), a differentiation marker for astrocytic cells. Using specific inhibitors and activators, we have shown that Ca2+, PKA, PI 3-K/PKB/GSK-3, Epac/Rap1/PP2A and MAP kinase modules are not involved in the inhibition of NPP1 gene expression. The transcription factor c-jun is significantly reduced while c-fos becomes up regulated after cAMP elevation. However an electrophoretic mobility shift assay with the activator protein-1 motif present in the promoter of the rat NPP1 gene indicates that this motif is not involved in the cAMP-dependent inhibition of NPP1 expression. In conclusion, these results indicate that intracellular cAMP levels regulate the expression of NPP1 in rat C6 glioma cells by a signalling pathway that is different from the GFAP signal transduction pathway. (C) 2010 Elsevier B.V. All rights reserved.