| Research Abstract |
In order to clarify the role of the phospholipase D (PLD) isozyme PLD1 and PLD2 in cell functions, PLD1 and PLD2 cDNA were transfected into various cells, and the influences on the signal transduction and cell functions were examined. At the early stages of apotosis induced by H_2O_2 or low oxygen in PC12 cell, PLD activity was increased transiently. The activation of PLD by H_2O_2 was attributed to PLD2, and was activated through the MAP kinase family (ERK and p38MAP kinase). Moreover, the apotosis induced by low oxygen was suppressed by transfection of PLD2 into PC12 cells, suggesting that PLD acts to prevent apotosis. It has been known that sphingosine 1-phosphate (S1P) is mitogen and participates in a cell survival. We examined a role of PLD in the survival signaling PI3 kinase and Akt using the CHO cell overexpressed with the S1P receptor EDG3. S1P stimulation of EDG3-CHO cells, but not vector-transfected cells, induced activation of PLD, PI3-kinase, and Akt. The Akt phosphorylati
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on was prevented by the PI3-kinase inhibitors wortmannin and LY294002, indicating that the Akt activation was dependent on PI3-kinase. S1P-induced activation of PI3-kinase and Akt was abrogated by 1-butanol, which inhibited S1P-induced accumulation of phosphatidic acid (PA) by PLD, whereas it was not inhibited by 2-butanol without such an action. Coexpression of the wild-type PLD2 with myc-Akt resulted in increased Akt activation in response to S1P. In contrast, co-expression of a catalytically inactive mutant of PLD2 eliminated the S1P-induced Akt activation. The treatment of EDG3-CHO cells with exogenous Streptomyces chromofuscus PLD, which caused an accumulation of PA, resulted in increases in PI 3-kinase activity and the phosphorylation of Akt, the latter of which was completely abolished by LY294002. Furthermore, SIP-induced membrane ruffling, which was dependent on PI 3-kinase and Rac, was inhibited by 1-butanol, but not 2-butanol. These results demonstrate that PLD participates in the activation of PI 3-kinase and Akt in stimulation of EDG3 receptor. Moreover, the remarkable high PLD2 activity was found in a human renal cancer. Immuno-stainining with the antibody of PLD2 revealed strong positive stain in the nuclei of a renal cancer compared with the normal tissue, suggesting that PLD2 may be associated with the participation in tumorigenesis. Less
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