Sphingosine-1-Phosphate Inhibition of Apoptosis Requires Mitogen-Activated Protein Kinase Phosphatase-1 in Mouse Fibroblast C3H10T½ Cells

Abstract

The roles of extracellular regulated kinase (ERK) activation and mitogen-activated protein kinase phosphatase-1 (MKP-1) were examined in sphingosine-1-phosphate (S1P)-mediated inhibition of apoptosis in C3H10T 1/2 fibroblast cells. Apoptosis induced by the ceramide analog, C8-ceramine, was inhibited by S1P (ceramine/S1P). Stress activated protein kinase or c-Jun N-terminal kinase (SAPK/JNK) activation was significantly higher after ceramine and ceramine/S1P treatments. Ceramine/S1P treatment also significantly increased ERK activation and MKP-1 protein levels. ERK activation was required for the inhibition of apoptosis by S1P as shown using the mitogen-activated protein kinase kinase inhibitor, PD98059. Transfection with a dominant negative mutant construct of the MKP-1 gene prevented S1P inhibition of apoptosis and resulted in sustained SAPK/JNK activity. The MKP-1 mutant did not affect ERK activity, indicating that MKP-1 preferentially down-regulates SAPK/JNK in C3H10T 1/2 cells. Finally, the S1P activation of ERK and inhibition of apoptosis were reduced by pertussis toxin treatment, suggesting that G-protein-coupled receptors, such as the endothelial differentiation gene (EDG) receptor, play a role. Thus, both ERK activation and MKP-1, which down-regulates SAPK/JNK, are required for S1P-mediated inhibition of apoptosis.