Effects of ceramide, ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2α; additional role of ceramide-1-phosphate in phosphorylation and Ca2+ signaling

Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A2 (PLA2) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca2+ binding regions in the C2 domain of α type cytosolic PLA2 (cPLA2α), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA2α has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4β-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca2+ ionophore (A23187) -induced release of AA via cPLA2α's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca2+ nor the phosphorylation of cPLA2α in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA2α that is independent of the Ca2+ signal and the PKC-ERK-mediated phosphorylation signal.