title: Oxidized low density lipoprotein-mediated activation of phospholipase D in smooth muscle cells: a possible role in cell proliferation and atherogenesis.
abstract: Low density lipoproteins (LDL) are risk factors in atherosclerosis and oxidative modification of LDL to oxidized LDL (OX-LDL) increases its atherogenicity. Development of atherosclerosis likely involves OX-LDL-mediated smooth muscle cell (SMC) proliferation. However, the mechanism(s) of SMC proliferation by OX-LDL is unknown. We hypothesized that OX-LDL may mediate SMC proliferation by activation of phospholipase D (PLD) through the generation of the second-messenger, phosphatidic acid (PA). To test this hypothesis, activation of PLD by OX-LDL was investigated in [3H]myristic acid- or [32P]orthophosphate-labeled rabbit femoral artery smooth muscle cells (RFASMC) in the presence of 0.5% ethanol or 0.05% butanol. Phospholipase D activation, as measured by labeled phosphatidylethanol (PEt) or phosphatidylbutanol (PBt) formation, was enhanced (3- to 5-fold) by OX-LDL. This activation of PLD was specific for OX-LDL, as native LDL or acetylated LDL had no effect. Further, OX-LDL-mediated [32P]PEt formation was dose- and time-dependent. To determine the mechanism(s) of OX-LDL-induced PLD activation, the role of protein kinase C (PKC) and Ca2+ was investigated. Pretreatment of [32P]orthophosphate-labeled RFASMC with known inhibitors of PKC such as staurosporine, calphostin-C, or H-7, had no effect on OX-LDL-induced PLD activation. Also, down-regulation of PKC by 12-O-tetradecanoylphorbol 13-acetate (TPA) (100 nM, 18 h) did not alter the OX-LDL-mediated [32P]PEt formation. However, pretreatment of RFASMC with genistein, a putative inhibitor of tyrosine kinases, attenuated the OX-LDL-mediated [32P]PEt formation. In addition, exposure of RFASMC to sodium orthovanadate, an inhibitor of phosphatases, enhanced the OX-LDL-mediated PLD activation. The effects of genistein and vanadate on PLD activation were specific for OX-LDL as these agents did not alter the TPA-induced [32P]PEt formation. Treatment of quiescent RFASMC with OX-LDL increased [3H]thymidine incorporation into DNA. This enhanced incorporation of [3H]thymidine into DNA was also mimicked by exogenously added phosphatidic acid (PA) or lysophosphatidic acid (LPA). These findings suggest that OX-LDL is a potent activator of the PLD pathway in SMC. The activation of PLD by OX-LDL generates second-messengers like PA and/or LPA which modulate mitogenesis. Thus, these results indicate that OX-LDL, in atherosclerotic lesions, may enhance SMC proliferation through the modulation of signal transduction pathways including activation of PLD.
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, USA.
authors.forename: V
authors.initials: V
authors.lastname: Natarajan
authors.forename: W M
authors.initials: WM
authors.lastname: Scribner
authors.forename: C M
authors.initials: CM
authors.lastname: Hart
authors.forename: S
authors.initials: S
authors.lastname: Parthasarathy
mesh_terms: D000818:Animals; D001161:Arteriosclerosis / Q000209:etiology*; D002118:Calcium / Q000378:metabolism; D002455:Cell Division; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005263:Femoral Artery; D019204:GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D015227:Lipid Peroxidation; D008077:Lipoproteins, LDL / Q000378:metabolism / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D010712:Phosphatidic Acids / Q000378:metabolism / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011817:Rabbits; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D004075:Diglycerides; D004791:Enzyme Inhibitors; D008077:Lipoproteins, LDL; D010712:Phosphatidic Acids; C032629:acetyl-LDL; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases; D010738:Type C Phospholipases; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D002118:Calcium