ABSTRACT
Apoptosis is a major mechanism for cell death in the nervous system during development. P2X(7) nucleotide receptors are ionotropic ATP receptors that mediate cell death under pathological conditions. We developed an in vitro protocol to investigate the expression and functional responses of P2X(7) nucleotide receptors during retinoic acid (RA)-induced neuronal differentiation of human SH-SY5Y neuroblastoma cells. Neuronal differentiation was examined measuring cellular growth arrest and neuritic processes elongation. We found that SH-SY5Y cells treated for 5 days with RA under low serum content exhibited a neuron-like phenotype with neurites extending more than twice the length of the cell body and cell growth arrest. Concurrently, we detected the abolishment of intracellular-free calcium mobilization and the down-regulation of P2X(7) nucleotide receptor protein expression that protected differentiated cells from neuronal cell death and reduced caspase-3 cleavage-induced by P2X(7) nucleotide receptor agonist. The role of P2X(7) nucleotide receptors in neuronal death was established by selectively antagonizing the receptor with KN-62 prior to its activation. We assessed the involvement of protein kinases and found that p38 signaling was activated in undifferentiated after nucleotide stimulation, but abolished by the differentiating RA pretreatment. Importantly, P2X(7) receptor-induced caspase-3 cleavage was blocked by the p38 protein kinase specific inhibitor PD169316. Taken together, our results suggest that RA treatment of human SH-SY5Y cells leads to decreased P2X(7) nucleotide receptor protein expression thus protecting differentiated cells from extracellular nucleotide-induced neuronal death, and p38 signaling pathway is critically involved in this protection of RA-differentiated cells.
Subject(s)
Neurons/drug effects , Receptors, Purinergic P2/genetics , Tretinoin/pharmacology , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Brain Neoplasms/physiopathology , Calcium Signaling/drug effects , Caspase 3/metabolism , Cell Death/drug effects , Cell Death/genetics , Cell Differentiation/drug effects , Cytoprotection/drug effects , Cytoprotection/genetics , Down-Regulation/drug effects , Down-Regulation/genetics , Gene Expression Regulation, Neoplastic/drug effects , HL-60 Cells , Humans , MAP Kinase Signaling System/physiology , Neuroblastoma/genetics , Neuroblastoma/pathology , Neuroblastoma/physiopathology , Neurons/metabolism , Neurons/pathology , Neurons/physiology , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2/physiology , Receptors, Purinergic P2X7 , Tumor Cells, Cultured , p38 Mitogen-Activated Protein Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/physiologyABSTRACT
Nucleotides released from cells due to stress, injury or inflammation, induce mitogenic effects in monocytes via activation of P2Y(2) nucleotide receptors (P2Y(2)Rs). Here we show that P2Y(2) nucleotide receptors in U937 monocytic cells regulate the activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) by inducing the clustering of alpha(v) integrins. The activation of phosphatidylinositol 3-kinase by P2Y(2)R ligands was required for alpha(v) clustering, suggesting a means whereby two different classes of receptors communicate to induce mitogenic responses in monocytic cells. P2Y(2)R-induced alpha(v) clustering was also associated with a flattened phenotype of the U937 cells, consistent with the role of the P2Y(2)R in regulating early events in cell migration.
