Stimulation of sphingosine-1-phosphate formation by the P2Y2 receptor in HL-60 cells:: Ca2+ requirement and implication in receptor-mediated Ca2+ mobilization, but not MAP kinase activation

Sphingosine-1-phosphate (SPP), produced by sphingosine kinase, has recently been reported to act as an intracellular second messenger for Ca2+ and mitogenic responses triggered by membrane receptors and as an extracellular ligand for specific SPP receptors. Here, we investigated the signaling pathway leading to SPP production by the G protein-coupled P2Y(2) receptor and its functional implication in human leukemia (HL-60) cells, which do not respond to extracellular SPP. P2Y(2) receptor activation by UTP or ATP resulted in rapid and transient production of SPP, which was insensitive to pertussis toxin and blocked by the sphingosine kinase inhibitor, DL-threo-dihydrosphingosine. Treatment of HL-60 cells with this inhibitor did not affect activation of mitogen-activated protein kinases, but suppressed Ca2+ mobilization by the P2Y(2) receptor. However, receptor-induced SPP production apparently required an increase in intracellular Ca2+ concentration, but not Ca2+ influx, and was mimicked by exposure of cells to Ca2+ ionophores. Taken together, activation of the P2Y(2) receptor stimulates SPP production in HL-60 cells, a process apparently not required for mitogen-activated protein kinase activation, but most likely representing an amplification system for receptor-mediated Ca2+ signaling.