Abstract

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 P2Y₂ receptor and its functional implication in human leukemia (HL-60) cells, which do not respond to extracellular SPP. P2Y₂ 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 P2Y₂ 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 P2Y₂ 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.