标题：Oxidized low density lipoprotein-mediated activation of phospholipase D in smooth muscle cells: a possible role in cell proliferation and atherogenesis.
出版社：American Society for Biochemistry and Molecular Biology
摘要：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.
