Nuclear ADP-ribosylation Factor (ARF)- and Oleate-dependent Phospholipase D (PLD) in Rat Liver Cells

Abstract

Two forms of phospholipase D (PLD) have been found to be present in nuclei isolated from rat hepatocytes by measuring phosphatidylbutanol produced from exogenous radiolabeled phosphatidylcholine in the presence of butanol. In nuclear lysates from either rat liver or ascites hepatoma AH 7974 cells, the PLD activity was markedly stimulated by a recombinant ADP-ribosylation factor (rARF) in the presence of the guanosine 5-O-(3-thiotriphosphate) (GTP␥S) and phosphatidylinositol 4,5-bisphosphate. ATP and phorbol-12-myristate 13-acetate had no synergistic effect on this PLD activity. On the other hand, the nuclear PLD was stimulated by unsaturated fatty acids, especially by oleic acid. The ARF-dependent nuclear PLD activity was increased in the S-phase of the regenerating rat liver after partial hepatectomy and also was much higher in AH 7974 cells than in the resting rat liver. In contrast, the levels of the oleate-dependent PLD activity remained constant throughout the cell cycle in liver regeneration. The intranuclear levels of the stimulating proteins of the nuclear PLD activity, e.g. ARF, RhoA, and protein kinase C␦ increased in the S-phase of the regenerating liver. These results suggested that the nuclear ARFdependent PLD activity may be associated with cell proliferation.It has been known that cell nuclei contain a variety of enzymes generating lipid second messengers, such as sphingomyelinase (1), phospholipase A2 (2), PI 1 -specific phospholipase C (3-6) and lipid kinases (7). Growth factors seem to be able to affect the phosphoinositide metabolism in nuclei, suggesting a subtle regulation during cell activation (8, 9). In addition, accumulation of diacylglycerol (DG) in nuclei and translocation of protein kinase C to them have been demonstrated in a variety of cell types (10, 11). A large rise in mass of DG, with only small changes in mass of phosphoinositides, suggested a source of DG other than polyphosphoinositides, for example, phosphatidylcholine (PC), in nuclei (12). A recent study (13) has demonstrated that phosphatidylethanol formation from phosphatidylcholine, which was specifically catalyzed by phospholipase D (PLD) in the presence of ethanol, was induced by PMA in nuclei isolated from kidney cells and that nuclei possess the ability to generate DG and phosphatidic acid through the PLD:phosphatidic acid phosphohydrolase pathway. Thus, upon cell stimulation with agonists, the enzymes involving PC metabolism are considered to be activated in the nucleus as well as in the plasma membrane.It was demonstrated that PLD activity can be regulated by several factors (14, 15); oleate, small G proteins (ARF and Rho family), phosphatidylinositol 4,5-bisphosphate (PIP 2 ), phosphatidylethanolamine (PE), Ca 2ϩ , protein kinase C, protein tyrosine kinase. Recently, two different types of PLD, oleatedependent and ARF-dependent, were isolated from rat brain membranes (16). The oleate-dependent PLD was purified from pig lung microsomes (17), and ARF-dependent PLD activity was found to be abundant in Golgi-enriched ...

Inhibition of Platelet-derived Growth Factor-induced Cell Growth Signaling by a Short Interfering RNA for EWS-Fli1 via Down-regulation of Phospholipase D2 in Ewing Sarcoma Cells

“…For example, enhanced expression and increased activity of two PLD isoforms have been found in cancer cells and tissues (33)(34)(35). We have observed that ADP-ribosylation factoractivated PLD activity was transiently increased in regenerating liver nuclei and that PLD2 levels were markedly elevated in the nuclei of hepatoma and kidney tumor cells (35,36). There is also evidence that PLD2 levels are markedly elevated in the caveolae of stimulated oncogenic cells and multidrugresistant cancer cells (37,38).…”

The Role of Phospholipase D in Regulated Exocytosis

“…The PM localization was reported to be consistent with high basal activity of the PLD2 isoform with no significant contribution from PLD1 (37). PLD1 was localized to the PM and intracellular vesicles in activated macrophages and PLD2 to the nucleus (59,60). Studies in rat embryo fibroblasts and COS-7 cells localized PLD1 to Golgi, endoplasmic reticulum, and secretory vesicles (56,58).…”

“…PLD1 was also found to be associated with caveolae-enriched microdomains (61). Therefore, there is no general consensus concerning the localization of PLD isoforms (59). Due to detection limits in assaying endogenous PA in the CV membrane, here we localize PLD activities by labeling CSC and CV with NBD-PC.…”

Oleic Acid and Angiotensin II Induce a Synergistic Mitogenic Response in Vascular Smooth Muscle Cells

“…This response may be mediated in part via other members of the mitogenactivated protein kinase family, eg, p38 and JNK. 36 -39 Oleic acid can potently stimulate nuclear phospholipase D. However, the nuclear ADP-ribosylation factor-dependent phospholipase D activity is associated with cell proliferation, 40,41 while oleate-dependent phospholipase D activitity is not.…”