RESUMO
We previously demonstrated that diperoxovanadate (DPV), a synthetic peroxovanadium compound and cell-permeable oxidant that acts as a protein tyrosine phosphatase inhibitor and insulinomimetic, increased phospholipase D (PLD) activation in endothelial cells (ECs). In this report, the regulation of DPV-induced PLD activation by mitogen-activated protein kinases (MAPKs) was investigated. DPV activated extracellular signal-regulated kinase, c-Jun NH2-terminal kinase (JNK), and p38 MAPK in a dose- and time-dependent fashion. Treatment of ECs with p38 MAPK inhibitors SB-203580 and SB-202190 or transient transfection with a p38 dominant negative mutant mitigated the PLD activation by DPV but not by phorbol ester. SB-202190 blocked DPV-mediated p38 MAPK activity as determined by activated transcription factor-2 phosphorylation. Immunoprecipitation of PLD from EC lysates with PLD1 and PLD2 antibodies revealed both PLD isoforms associated with p38 MAPK. Similarly, PLD1 and PLD2 were detected in p38 immunoprecipitates from control and DPV-challenged ECs. Binding assays demonstrated interaction of glutathione S-transferase-p38 fusion protein with PLD1 and PLD2. Both PLD1 and PLD2 were phosphorylated by p38 MAPK in vitro, and DPV increased phosphorylation of PLD1 and PLD2 in vivo. However, phosphorylation of PLD by p38 failed to affect PLD activity in vitro. These results provide evidence for p38 MAPK-mediated regulation of PLD in ECs.
Assuntos
Endotélio Vascular/enzimologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Peróxidos/farmacologia , Fosfolipase D/metabolismo , Vanadatos/farmacologia , Animais , Bovinos , Células Cultivadas , Endotélio Vascular/citologia , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Genes Dominantes , Humanos , Imidazóis/farmacologia , Isoenzimas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação/genética , Mutação/fisiologia , Fosforilação , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Reactive oxygen species (ROS) are implicated in the pathophysiology of several vascular disorders including atherosclerosis. Although the mechanism(s) of ROS-induced vascular damage remains unclear, there is increasing evidence for ROS-mediated modulation of signal transduction pathways. Exposure of bovine pulmonary artery endothelial cells to hydrogen peroxide (H(2)O(2)) enhanced tyrosine phosphorylation of 60- to 80- and 110- to 130-kDa cellular proteins, which were determined by immunoprecipitation with specific antibodies focal adhesion kinase (p125(FAK)) and paxillin (p68). Brief exposure of cells to a relatively high concentration of H(2)O(2) (1 mM) resulted in a time- and dose-dependent tyrosine phosphorylation of FAK, which reached maximum levels within 10 min (290% of basal levels). Cytoskeletal reorganization as evidenced by the appearance of actin stress fibers preceded H(2)O(2)-induced tyrosine phosphorylation of FAK, and the microfilament disruptor cytochalasin D also attenuated the tyrosine phosphorylation of FAK. Treatment of BPAECs with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid-AM attenuated H(2)O(2)-induced increases in intracellular Ca(2+) but did not show any consistent effect on H(2)O(2)-induced tyrosine phosphorylation of FAK. Several tyrosine kinase inhibitors, including genistein, herbimycin, and tyrphostin, had no detectable effect on tyrosine phosphorylation of FAK but attenuated the H(2)O(2)-induction of mitogen-activated protein kinase activity. We conclude that H(2)O(2)-induced increases in FAK tyrosine phosphorylation may be important in H(2)O(2)-mediated endothelial cell activation.
Assuntos
Moléculas de Adesão Celular/metabolismo , Endotélio Vascular/enzimologia , Peróxido de Hidrogênio/farmacologia , Proteínas Tirosina Quinases/metabolismo , Tirosina/metabolismo , Actinas/fisiologia , Animais , Cálcio/farmacologia , Bovinos , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Resistência a Medicamentos , Endotélio Vascular/citologia , Inibidores Enzimáticos/farmacologia , Proteína-Tirosina Quinases de Adesão Focal , Paxilina , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/fisiologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Artéria Pulmonar/citologia , Artéria Pulmonar/enzimologiaRESUMO
Reactive oxygen species (ROS) are implicated in the pathophysiology of a number of vascular disorders, including atherosclerosis. Recent studies indicate that ROS modulate signal transduction in mammalian cells. Previously, we have shown that ROS (hydrogen peroxide, fatty acid hydroperoxide, diperoxovanadate, and 4-hydroxynonenal) enhance protein tyrosine phosphorylation and activate phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). In the present study, our aim was to investigate the role of exogenous thiol agents on ROS-induced PLD activation in conjunction with the role of cellular thiols--glutathione (GSH) and protein thiols--on PLD activation and protein tyrosine phosphorylation. Pretreatment of BPAECs with N-acetyl-L-cysteine (NAC) or 2-mercaptopropionylglycine (MPG) blocked ROS-induced changes in intracellular GSH and PLD activation. Also, pretreatment with NAC attenuated diperoxovanadate-induced protein tyrosine phosphorylation. Pretreatment of BPAECs with diamide or L-buthionine-(S,R)-sulfoximine (BSO), agents that lower intracellular GSH and thiols, enhanced PLD activity. Furthermore, NAC blocked diamide- or BSO-mediated changes in GSH levels, PLD activity, and protein tyrosine phosphorylation. NAC also attenuated diamide-induced tyrosine phosphorylation of proteins between 69 and 118 KDa. These results support the hypothesis that modulation of thiol-redox status (cellular nonprotein and protein thiols) may contribute to the regulation of ROS-induced protein tyrosine phosphorylation and PLD activation in vascular endothelium.
Assuntos
Antioxidantes/metabolismo , Endotélio Vascular/enzimologia , Fosfolipase D/metabolismo , Tioureia/análogos & derivados , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Butionina Sulfoximina/farmacologia , Bovinos , Linhagem Celular , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Líquido Intracelular/metabolismo , Oxirredução , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Artéria Pulmonar , Espécies Reativas de Oxigênio/fisiologia , Compostos de Sulfidrila/farmacologia , Reagentes de Sulfidrila/farmacologia , Tioureia/farmacologia , Tiopronina/farmacologiaRESUMO
Tyrosine phosphorylation of proteins, controlled by tyrosine kinases and protein tyrosine phosphatases, plays a key role in cellular growth and differentiating. A wide variety of hormones, growth factors, and cytokines modulate cellular tyrosine phosphorylation to transmit signals across the plasma membrane to the nucleus. Recent studies suggest that reactive oxygen species (ROS) also induce cellular protein tyrosine phosphorylation through receptor or nonreceptor tyrosine kinases. To determine whether protein tyrosine phosphorylation by ROS regulates endothelial cell (EC) metabolism and function, we exposed vascular ECs to H2O2 or H2O2 plus vanadate. This resulted in a time- and dose-dependent increase in protein tyrosine phosphorylation of several proteins (M(r) 21-200 kDa), as determined by immunoprecipitation and Western blot analysis with antiphosphotyrosine antibody. Immunoprecipitation with specific antibodies identified increased tyrosine phosphorylation of mitogen-activated protein kinases (42-44 kDa), paxillin (68 kDa), and FAK (125 kDa) by ROS. An immediate signaling response to increased protein tyrosine phosphorylation by ROS was activation of phospholipases such as A2, C, and D. Suramin pretreatment inhibited ROS stimulation of phospholipase D (PLD), suggesting a role for growth factor receptors in this activation. Further, PLD activation by ROS was attenuated by N-acetylcysteine, indicating that intracellular thiol status is critical to ROS-mediated signal transduction. These results provide evidence that ROS modulate EC signal transduction via a protein tyrosine phosphorylation-dependent mechanism.
Assuntos
Endotélio Vascular/metabolismo , Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tirosina/metabolismo , Animais , Antioxidantes/farmacologia , Cálcio/metabolismo , Bovinos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peróxidos/toxicidade , Fosfolipases/metabolismo , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Vanadatos/toxicidadeRESUMO
Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [32P] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by Western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [32P] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC alpha and epsilon, the major isotypes of PKC in BPAECs, by TPA (100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [32P] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.
Assuntos
Cálcio/fisiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Peróxidos/farmacologia , Fosfolipase D/metabolismo , Proteínas Tirosina Quinases/metabolismo , Vanadatos/farmacologia , Animais , Bovinos , Linhagem Celular , Endotélio Vascular/citologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Líquido Intracelular/química , Oxirredução/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Artéria Pulmonar/citologia , Espécies Reativas de Oxigênio , Compostos de Sulfidrila/farmacologiaRESUMO
We have previously reported that endothelial cell phospholipase D (PLD), activated by 4-hydroxynonenal (4-HNE), was independent of protein kinase C activation. To determine whether PLD stimulation by 4-HNE is related to protein tyrosine phosphorylation, the effects of tyrosine kinase (Tyrk) and protein tyrosine phosphatase (PTPase) inhibitors on PLD activation were investigated. Pretreatment of bovine pulmonary artery endothelial cells (BPAEC) with Tyrk inhibitors, such as genistein, erbstatin, and herbimycin attenuated 4-HNE-induced PLD activation. Furthermore, vanadate, phenylarsine oxide, and diamide, inhibitors of PTPases, markedly increased the 4-HNE-induced PLD activation. The effects of Tyrk and PTPase inhibitors were specific towards the 4-HNE, as these agents had no effect on the agonist- or TPA-induced PLD activation. In addition to PLD activation, treatment of BPAEC with 4-HNE increased tyrosine phosphorylation of proteins including bands of molecular weights 40,000-60,000, 70,000-90,000, and 110,000-130,000. The 4-HNE-mediated increase in protein tyrosine phosphorylation was partly inhibited by genistein (100 microM). Vanadate (10 microM) pretreatment also potentiated 4-HNE-induced protein tyrosine phosphorylation. These data suggest that 4-HNE-mediated stimulation of PLD may occur as a result of activation of tyrosine kinases.
Assuntos
Aldeídos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fosfolipase D/metabolismo , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Animais , Bradicinina/farmacologia , Bovinos , Células Cultivadas , Sinergismo Farmacológico , Endotélio Vascular/enzimologia , Ativação Enzimática , Genisteína , Ionomicina/farmacologia , Isoflavonas/farmacologiaRESUMO
Naturally occurring polycations and cationic proteins are implicated in vascular disorders. It is known that activated leukocytes and platelets release polycations, such as polylysine (PLys), of varying molecular sizes into the vasculature, and some of these have been described to be bactericidal. Polycations interact with endothelial cells (ECs) and cause alterations in permeability and cellular functions. The precise mechanism(s) by which polycations bring about cellular changes is unknown. Here, we report that the polycations PLys and polyarginine (PArg) induce phospholipase D (PLD) activation in ECs. Polycation-mediated PLD activation was both time and concentration dependent, and activation of PLD was not due to cytotoxicity. PArg was more potent compared with PLys of the same molecular weight in stimulation of PLD. Treatment with bisindolylmaleimide, a specific protein kinase C (PKC) inhibitor, and heparin attenuated polycation-mediated PLD activation. Furthermore, downregulation of PKC by 12-O-tetradecanoylphorbol-13-acetate (100 nM, 18 h) also blocked polycation-mediated PLD stimulation. These data suggest that polycation-mediated PLD stimulation probably involves PKC and may represent an important cellular response to leukocyte/platelet activation in the vascular endothelium.
Assuntos
Cátions/farmacologia , Endotélio Vascular/enzimologia , Células 3T3/enzimologia , Animais , Bovinos , Células Cultivadas , Endotélio Vascular/citologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Fibroblastos/enzimologia , L-Lactato Desidrogenase/metabolismo , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/enzimologia , Peptídeos/farmacologia , Polilisina/farmacologia , Inibidores de Proteínas Quinases , Artéria Pulmonar/citologia , Artéria Pulmonar/enzimologiaRESUMO
Phosphatidic acid generation by phospholipase D (PLD) activation has been implicated in agonist- and oxidant-mediated endothelial cell signal transduction. We examined the effect of wortmannin on PLD activation in pulmonary artery endothelial and smooth muscle cells in culture. Pretreatment of bovine pulmonary artery endothelial cells (BPAECs) with wortmannin potentiated TPA- (100 nM), ATP- (100 microM), and bradykinin- (1 microM) induced [32P]PEt formation, an index of PLD activation. However, wortmannin by itself had no effect on PLD activity. The potentiating effect of wortmannin on TPA-induced PLD activation was dose- (1-10 microM) and time-dependent (5-30 min) and was inhibited by bisindoylmalemide, an inhibitor of protein kinase C (PKC). Furthermore, down-regulation of PKC by prolonged treatment with TPA (100 nM, 18 h) attenuated the wortmannin effect. This effect of wortmannin was specific for TPA- or agonist-induced PLD activation as no potentiation of [32P]PEt formation was observed with H2O2 (1 mM) or ionomycin (1 microM). The effect of wortmannin was not due to activation of PKC alpha as determined by western blot analysis of PKC alpha in the cytosol and membrane fractions. Also, genistein, an inhibitor of tyrosine kinases, did not attenuate the wortmannin-mediated potentiation of PLD thereby suggesting non-involvement of protein tyrosine phosphorylation. These results indicate that wortmannin potentiates PKC-dependent stimulation of PLD in vascular endothelial cells.
Assuntos
Androstadienos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Glicerofosfolipídeos , Inibidores de Fosfodiesterase/farmacologia , Fosfolipase D/metabolismo , Proteína Quinase C/fisiologia , Animais , Bovinos , Células Cultivadas , Cromonas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Genisteína , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Isoflavonas/farmacologia , Morfolinas/farmacologia , Oxidantes/farmacologia , Ácidos Fosfatídicos/biossíntese , Radioisótopos de Fósforo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Sensibilidade e Especificidade , Acetato de Tetradecanoilforbol/farmacologia , WortmaninaRESUMO
Oxidants play a significant role in endothelial cell dysfunction through modulation of diverse biochemical reactions and signal transduction pathways. Towards understanding the role of oxidants in vascular injury, we studied the effect of hydrogen peroxide (H2O2), vanadate, and pervanadate (V(4+)-OOH) on [32Pi] uptake and protein phosphorylation in bovine pulmonary artery endothelial cells (BPAEC). The incorporation of labelled [32Pi] into BPAEC was dependent on the concentration of the oxidant employed and time of incubation. Of the oxidants tested, pervanadate (10 microM) induced maximum incorporation of [32Pi] into cells (two- to threefold over control) followed by H2O2 (1 mM) and vanadate (100 microM) and clear differences in labeled protein profiles were noticed between control and oxidant treated cells. The proteins, analyzed by SDS-PAGE, showed distinct increases in labeling patterns ranging from 21-205 kDa, as evidenced by autoradiography. While the majority of the incorporated [32Pi] was in serine/threonine residues, immunoprecipitation and immunoblotting of cell lysates, using an antiphosphotyrosine antibody, revealed that oxidant treatment resulted in significant increases in total protein tyrosine phosphorylation. Most significantly, immunoprecipitation of cell lysates, from pervanadate treatment showed distinct tyrosine phosphorylation of 22 kDa protein, which was identified as caveolin, a marker of caveolae. Pervanadate-mediated phosphorylation was effectively inhibited by staurosporine (5 microM), while genistein showed only partial attenuation. Furthermore, H2O2 treatment resulted in enhanced phosphorylation of 24 kDa protein, which was attenuated by genistein. In addition, oxidant-treated cells exhibited increased tyrosine kinase activity and decreased phosphatase activity. These data show differences in labeling profiles of proteins in response to different oxidants, suggesting differential modulation of distinct protein kinases/phosphatases.
Assuntos
Caveolinas , Endotélio Vascular/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Oxidantes/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Animais , Bovinos , Caveolina 1 , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/farmacologia , Fosforilação , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Vanadatos/farmacologiaRESUMO
Small cell lung carcinoma (SCLC) frequently metastasizes early in the course of the disease. P-selectin (P-sel), a cell adhesion molecule expressed on activated platelets and endothelial cells (EC), has previously been demonstrated to mediate binding of platelets to SCLC. We hypothesized that P-sel facilitates attachment of SCLC to EC, acting as an important factor in SCLC metastasis. To test this hypothesis, attachment of H82 cells (SCLC cell line) to EC was quantified. Attachment of H82 cells to 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated EC was increased compared with control EC. Increased attachment of H82 cells to EC was apparent after 10 min of TPA activation, reached a peak after 30 min, and returned to baseline after 120 min of exposure. The TPA-induced increase in H82 cell attachment to EC was inhibited by addition of anti-P-sel antibodies but not by addition of anti-E-selectin antibodies. The TPA-induced increase in H82 cell attachment was likely mediated by activation of EC protein kinase C (PKC). Pretreatment of the EC with PKC inhibitors effectively blocked the TPA-mediated increase in H82 cell attachment. In addition, prolonged exposure of EC to TPA resulted in decreased expression of the PKC-alpha and PKC-epsilon isoforms. These data indicate for the first time that attachment of SCLC to activated EC appears to be mediated by increased expression of P-sel on the EC surface, which may result from activation of specific isoforms of PKC.
Assuntos
Carcinoma de Células Pequenas/patologia , Endotélio Vascular/patologia , Neoplasias Pulmonares/patologia , Selectina-P/metabolismo , Animais , Carcinógenos/farmacologia , Bovinos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Endotélio Vascular/metabolismo , Humanos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Oxidant-induced activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAEC) is independent of protein kinase C and calcium. In the present study, the effects of tyrosine kinase and protein tyrosine phosphatase (PTPase) inhibitors on hydrogen peroxide (H2O2)-induced PLD activation and protein tyrosine phosphorylation were examined in BPAEC. Pretreatment of BPAEC with putative tyrosine kinase inhibitors genistein, tyrphostin, and herbimycin attenuated H2O2 (1 mM)-induced PLD activation. The inhibitory effect of the tyrosine kinase inhibitors was highly specific for H2O2-induced modulation and showed no effect on PLD activation mediated by 12-O-tetradecanoylphorbol 13-acetate or bradykinin. Furthermore, addition of H2O2 increased in a time-dependent manner tyrosine phosphorylation of several proteins (17-200 kDa), as determined by immunoblot analysis with antiphosphotyrosine antibodies. H2O2-mediated protein tyrosine phosphorylation preceded PLD activation, and a good correlation was observed on the effect of genistein in H2O2-induced PLD activation and protein tyrosine phosphorylation. Addition of vanadate, a phosphotyrosine phosphatase inhibitor, synergistically increased both PLD activation and protein tyrosine phosphorylation mediated by H2O2. Moreover, vanadate by itself had minimal effect on basal PLD activity in BPAEC; however, at 10 microM vanadate, an increase in protein tyrosine phosphorylation was observed. In addition to vanadate, phenylarsine oxide and diamide potentiated H2O2-induced PLD activation. These results suggest that tyrosine kinase activation may be involved in H2O2-induced PLD activation in vascular endothelial cells.
Assuntos
Endotélio Vascular/metabolismo , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Fosfolipase D/metabolismo , Tirosina/metabolismo , Animais , Bovinos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Fosforilação , Transdução de Sinais/efeitos dos fármacosRESUMO
Activation of phospholipase D (PLD) represents part of an important signalling pathway in mammalian cells. Phospholipase D catalyzed hydrolysis of phospholipids generates phosphatidic acid (PA) which is subsequently metabolized to lyso-PA (LPA) or diacylglycerol (DAG). While DAG is an endogenous activator of protein kinase C (PKC), PA and LPA have been recognized as second messengers as well. Activation of PLD in response to an external stimulus may involve PKC, Ca2+, G-proteins and/or tyrosine kinases. In this review, we will address the role of protein tyrosine phosphorylation in growth factor-, agonist- and oxidant-mediated activation of PLD. Furthermore, a possible link between PKC, Ca2+, G-proteins and tyrosine kinases is discussed to indicate the complexity involved in the regulation of PLD in mammalian cells.
Assuntos
Fosfolipase D/fisiologia , Proteínas Tirosina Quinases/fisiologia , Animais , Ativação Enzimática , Humanos , Fosfolipase D/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sistemas do Segundo Mensageiro/fisiologiaRESUMO
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.
Assuntos
Arteriosclerose/etiologia , Divisão Celular , Peroxidação de Lipídeos , Lipoproteínas LDL/farmacologia , Músculo Liso Vascular/enzimologia , Fosfolipase D/metabolismo , Animais , Cálcio/metabolismo , Diglicerídeos/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Artéria Femoral , Proteínas de Ligação ao GTP/fisiologia , Humanos , Lipoproteínas LDL/metabolismo , Ácidos Fosfatídicos/metabolismo , Ácidos Fosfatídicos/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Coelhos , Fosfolipases Tipo C/metabolismoRESUMO
We have investigated the activation of phospholipase D (PLD) by sphingosine and its derivatives in bovine pulmonary artery endothelial cells (BPAEC) prelabeled with [32P]orthophosphate or [32P]lyso phospholipids. Sphingosine, in a dose- and time-dependent manner, stimulated the hydrolysis of [32P]phosphatidylcholine (PC) resulting in the production of [32P]phosphatidic acid (PA), suggesting PLD activation. In the presence of ethanol (150 mM), the accumulation of [32P]phosphatidylethanol was also observed. The sphingosine-induced stimulation of PLD activity was not affected by treatment with the protein kinase C (PKC) inhibitor staurosporine or by down-regulation of PKC with TPA and was independent of extracellular Ca2+, suggesting that the PLD activation was independent of PKC and Ca2+. Chelation of intracellular Ca2+ with BAPTA actually potentiated the sphingosine-stimulated [32P]PC hydrolysis. Furthermore, the activation of PLD by sphingosine was not abolished by treatment of BPAEC with either cholera or pertussis toxin, indicating noninvolvement of toxin-sensitive G-proteins. In addition to hydrolysis of [32P]PC, sphingosine also stimulated PLD-mediated hydrolysis of [32P]phosphatidylethanolamine and [32P]phosphatidylinositol. Among the various sphingoid compounds, in addition to sphingosine, only sphingosine-1-phosphate (Sph-1-P) activated the endothelial cell PLD. The effect of sphingosine and Sph-1-P on PA phosphatase (PA Pase) activity was tested using [3H]glycerol-labeled PA. The Mg(2+)-independent and membrane-associated PA Pase activity was inhibited by sphingosine (IC50 = 200 microM) but not by Sph-1-P. This implies that sphingosine and Sph-1-P share a similar PLD-stimulating property but differ in their PA Pase inhibitory activity.
Assuntos
Endotélio Vascular/enzimologia , Fosfolipase D/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Animais , Cálcio/metabolismo , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Desoxiglucose/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Ativação Enzimática , Cinética , Lisofosfolipídeos/metabolismo , Fosfatos/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfolipídeos/biossíntese , Fosfolipídeos/metabolismo , Radioisótopos de Fósforo , Proteína Quinase C/metabolismo , Artéria Pulmonar , Ribonucleotídeos/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
We have examined the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAEC) treated with 4-hydroxynonenal (4-HNE). Treatment of BPAEC labelled with [32P] orthophosphate (5 h for minimal phospholipid labelling) and [3H] myristic acid (24 h) with 4-HNE in the presence of 0.5% ethanol resulted in the formation of [3H] phosphatidylethanol (PEt) and [3H] phosphatidic acid (PA) with very little accumulation of [32P] PEt. The formation of [3H] PEt, as opposed to [32P] PEt, suggests that PEt synthesis was not through de novo pathway but rather through the PLD mechanism. 4-Hydroxynonenal-induced PLD activation was dose and time dependent, and was not associated with cytotoxicity as determined by [3H] deoxyglucose release. The formation of PEt was not affected by chelation of either extracellular Ca2+ with EGTA (5 mM, 30 min) or intracellular Ca2+ with BAPTA-AM (25 microM, 30 min). Treatment of BPAEC with either staurosporine (10 microM, 15 min), a protein kinase C (PKC) inhibitor, or down regulation of PKC by chronic 12-0-tetradecanoylphorbol-13-acetate (TPA) treatment (100 nM, 18 h) had no effect on 4-HNE-induced PLD activation. These results indicate that PLD activation by 4-HNE is independent of PKC activity. We also examined the specificity of nonylaldehyde derivatives and hydroxyalkenals on PLD activation. In addition to 4-HNE, 4-hydroxyoctenal and 4-hydroxyhexenal also stimulated [32P] PEt formation. Among the various nonylaldehydes examined, only trans-2-nonenal and trans-2-cis 6-nonadienal exhibited PLD activation, suggesting the requirement of a trans double bond at carbon 2 and a hydroxyl group at carbon 4. However, in contrast to 4-HNE-induced PLD activation of BPAEC monolayers, treatment of 105,000 x g membranes with 4-HNE had no effect on PLD catalyzed hydrolysis of [2-14C] oleoyl phosphatidylcholine. These data provide evidence that 4-HNE, a metabolite of membrane lipid peroxidation, may be involved in endothelial cell signal transduction, through the activation of phospholipase D and the generation of second messengers like phosphatidic acid and diacylglycerol.