The metabotropic P2Y ₄ receptor participates in the commitment to differentiation and cell death of human neuroblastoma SH-SY5Y cells

Extracellular nucleotides exert a variety of biological actions through different subtypes of P2 receptors. Here we characterized in the human neuroblastoma SH-SY5Y cells the simultaneous presence of various P2 receptors, belonging to the P2X ionotropic and P2Y metabotropic families. Western blot analysis detected the P2X _1,2,4,5,6,7 and P2Y _1,2,4,6, but not the P2X ₃ and P2Y ₁₂ receptors. We then investigated which biological effects were mediated by the P2Y ₄ subtype and its physiological pyrimidine agonist UTP. We found that neuronal differentiation of the SH-SY5Y cells with dibutiryl-cAMP increased the expression of the P2Y ₄ protein and that UTP itself was able to positively interfere with neuritogenesis. Moreover, transient transfection and activation of P2Y ₄ also facilitated neuritogenesis in SH-SY5Y cells, as detected by morphological phase contrast analysis and confocal examination of neurofilament proteins NFL. This was concurrent with increased transcription of immediate-early genes linked to differentiation such as cdk-5 and NeuroD6, and activity of AP-1 transcription family members such as c-fos, fos-B, and jun-D. Nevertheless, a prolonged activation of the P2Y ₄ receptor by UTP also induced cell death, both in naive, differentiated, and P2Y ₄- transfected SH-SY5Y cells, as measured by direct count of intact nuclei and cytofluorimetric analysis of damaged DNA. Taken together, our data indicate that the high expression and activation of the P2Y ₄ receptor participates in the neuronal differentiation and commitment to death of SH-SY5Y cells.