| Research Abstract |
Sphingosine kinase (SPHK) has been known to be important signaling enzyme to produce sphingosine 1-phosphate (S1P) which is involved in a variety of cell functions through specific receptor EDG family. Two type of SPHK (1,II) have been cloned and novel type of SPHK is present in human platelets. We prepared a rabbit polyclonal antibody against the C-terminal oligopeptide of SPHK1a. Using this anti-SPHK1a antibody, the cell type-specificlocalization of SPHK1a in human tissues was analyzed immunohistochemically. Strong positice staining for SPHK1a was observed in the white matter in the cerebrum and cerebellum, the uriniferous tubules in the kidney, in megakaryocytesand platelets. S1P stimulation of EDG3-overexpressing CHO cells, but not vector-transfected cells, induced activation of PLD, PI 3-kinase, and Akt. S1P-induced activation of PI 3-kinase and Akt was abrogated by 1-butanol, which inhibited S1P-induced accumulation of phosphatidic acid (PA) by PLD. Coexpression of the wild-type PLD2 with myc-Aktresulted in increased Akt activationin response to S1P. These results demonstrate that PLD participatesin the activation of PI 3-kinase and Akt in S1P-stimulation of EDG3 receptor. We demonstrated that TNF-α induced SPHK activationand increase of S1P in human hepatocytes and dimethylsphingosine, a SPHK inhibitor, inhibited PI3K/Akt pathway and potentiated TNF-α-mediated hepatocyteapoptosis. These results suggest that SPHK and S1P play a role in cell survival through activation of PI3K/Akt signaling pathway.
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