Oxidative stress has been implicated in mediation of vascular disorders. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD1, protein kinase C-a (PKC-a), and other unidentified proteins in rat vascular smooth muscle cells (VSMCs). Interestingly, PLD1 was found to be constitutively associated with PKC-a in VSMCs. Stimulation of the cells by H2O2 and vanadate showed a concentration-dependent tyrosine phosphorylation of the proteins in PLD1 immunoprecipitates and activation of PLD. Pretreatment of the cells with the protein tyrosine kinase inhibitor, genistein resulted in a dose-dependent inhibition of H2O2-induced PLD activation. PKC inhibitor and down-regulation of PKC abolished H2O2-stimulated PLD activation. The cells stimulated by oxidative stress (H2O2) caused increased cell migration. This effect was prevented by the pretreatment of cells with tyrosine kinase inhibitors, PKC inhibitors, and 1-butanol, but not 3-butanol. Taken together, these results suggest that PLD might be involved in oxidative stress-induced migration of VSMCs, possibly via tyrosine phosphorylation and PKC activation.
Animals ; Cell Movement/drug effects/*physiology ; Cells, Cultured ; Enzyme Activation/drug effects ; Enzyme Inhibitors/pharmacology ; Genistein/pharmacology ; Hydrogen Peroxide/pharmacology ; Muscle, Smooth, Vascular/cytology/*physiology ; *Oxidative Stress/drug effects ; Phospholipase D/*metabolism ; Phosphorylation/drug effects ; Protein Kinase C/*metabolism ; Protein-Tyrosine Kinase/antagonists & inhibitors ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Signal Transduction/drug effects ; Vanadates/pharmacology ; Vascular Diseases/metabolism

Hepatitis C Virus (HCV) is associated with a severe liver disease and increased frequency in the development of hepatocellular carcinoma. Overexpression of HCV core protein is known to transform fibroblast cells. Phospholipase D (PLD) activity is commonly elevated in response to mitogenic signals, and has also been overexpressed and hyperactivated in some human cancer cells. The aim of this study was to understand how PLD was regulated in the HCV core protein-transformed NIH3T3 mouse fibroblast cells. We observed that PLD activity was elevated in the NIH3T3 cells overexpressing HCV core protein over the vector alone-transfected control cells, however, expression levels of PLD protein and protein kinase C (PKC) in the HCV core protein-transformed cells was similar to the control cells. Phorbol 12-myristate 13-acetate (PMA), which is known to activate PKC, stimulated PLD activity significantly more in the core protein-transformed cells, in comparison with that of the control cells. PLD activity assay using PKC isozyme-specific inhibitor and PKC translocation experiment showed that PKC-delta was mainly involved in the PMA- induced PLD activation in the core-transformed cells. Moreover, in cells overexpressing HCV core protein, PMA also stimulated p38 kinase more potently than that of the control cells, and an inhibitor of p38 kinase abolished PMA-induced PLD activation in cells overexpressing HCV core protein. Taken together, these results suggest that PLD might be implicated in core protein-induced transformation.
Animals ; Cell Line, Transformed ; *Cell Transformation, Viral ; Fibroblasts/enzymology/virology ; Hepacivirus/genetics/*physiology ; Mice ; NIH 3T3 Cells ; Phospholipase D/*metabolism ; Protein Kinase C/antagonists & inhibitors/physiology ; Protein Transport/drug effects ; Research Support, Non-U.S. Gov't ; Tetradecanoylphorbol Acetate/*analogs & derivatives/pharmacology ; Transfection ; Up-Regulation ; Viral Core Proteins/genetics/*metabolism ; p38 Mitogen-Activated Protein Kinases/physiology