RESUMEN
Certain proteins, including receptors and signaling molecules, are known to be enriched in caveolae and lipid rafts. Caveolin-1, the major structural protein of caveolae, specifically interacts with many signaling molecules and, thus, caveolae and lipid rafts are often seen as preassembled signaling platforms. A potential binding site for caveolin-1 is present in the platelet-activating factor receptor (PAFR) sequence, and many downstream signaling components of PAFR activation preferentially localize in caveolae. The aim of this study was to investigate whether the PAFR was localized in caveolae/lipid raft domains and, if so, what would be the significance of such localization for PAFR signaling. In this study, we demonstrate that PAFR localizes within membrane microdomains, in close proximity to caveolin-1 in living cells, with potential interaction through a caveolin-1-binding sequence in the PAFR C terminus. Caveolin-1, however, is not essential for PAFR localization in lipid rafts. Disruption of caveolae/lipid rafts with methyl-beta-cyclodextrin markedly reduced PAF-triggered inositol phosphate production and cytosolic calcium flux, suggesting that PAFR signaling through the Galphaq protein was critically dependent on integrity of lipid rafts and/or caveolae. Interestingly, whereas in caveolin-1-expressing cells lipid raft disruption markedly decreased PAFR-mediated activation of the ERK/MAPK pathway, in cells lacking caveolae, such as leukocytes, lipid raft disruption had either the same inhibitory effect (Ramos B cells) or no effect (monocytes) on PAFR capacity to signal through the ERK/MAPK pathway. In conclusion, PAFR appears to localize within caveolae or lipid rafts in different cell types, and this location may be important for specific signaling events.
Asunto(s)
Señalización del Calcio , Caveolas/metabolismo , Caveolina 1/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor de Activación Plaquetaria/fisiología , Animales , Sitios de Unión/inmunología , Células CHO , Señalización del Calcio/inmunología , Caveolas/enzimología , Caveolas/inmunología , Línea Celular , Línea Celular Tumoral , Cricetinae , Cricetulus , Activación Enzimática/inmunología , Humanos , Microdominios de Membrana/inmunología , Microdominios de Membrana/metabolismo , Fragmentos de Péptidos/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologíaRESUMEN
Platelet-activating factor (PAF) is a potent phospholipid mediator involved in several diseases such as allergic asthma, atherosclerosis and psoriasis. The human PAF receptor (PAFR) is a member of the G-protein-coupled receptor family. Following stimulation, PAFR becomes rapidly desensitized; this refractory state is dependent on PAFR phosphorylation, internalization and down-regulation. In this report, we show that the PAFR inverse agonist, WEB2086, can induce phosphorylation and down-regulation of PAFR. Using selective inhibitors, we determined that the agonist, PAF, and WEB2086 could induce phosphorylation of PAFR by PKC. Moreover, dominant-negative (DN) mutant of PKC isoforms beta inhibited WEB2086-stimulated PAFR phosphorylation, whereas PAF-stimulated phosphorylation was inhibited by DN PKCalpha and delta. WEB2086 also induced PAFR down-regulation which could be blocked by PKC inhibitors and by DN PKCbeta. WEB2086-induced down-regulation was dynamin-dependent but arrestin-independent. Unlike PAF, WEB2086-stimulated intracellular trafficking of PAFR was independent of Rab5. Specific inhibitors of lysosomal proteases and of proteasomes were both effective in reducing WEB2086-induced PAFR down-regulation, indicating the importance of receptor targeting to both lysosomes and proteasomes in long-term cell desensitization to WEB2086. These results indicate that although both agonists and inverse agonists induce receptor PAFR down-regulation, this may be accomplished through different signal transduction and trafficking pathways.
Asunto(s)
Azepinas/farmacología , Regulación hacia Abajo , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Transducción de Señal , Triazoles/farmacología , Animales , Células COS , Chlorocebus aethiops , Lisosomas/efectos de los fármacos , Factor de Activación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/agonistas , Glicoproteínas de Membrana Plaquetaria/metabolismo , Inhibidores de Proteasoma , Proteína Quinasa C/metabolismo , Transporte de Proteínas/efectos de los fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
The acute increase in vascular permeability produced by vascular endothelial growth factor (VEGF-A(165)) requires activation of endothelial Flk-1 receptors (VEGFR-2) and stimulation of platelet-activating factor (PAF) synthesis. Like PAF, VEGF-A(165) promotes translocation of P-selectin to the endothelial cell (EC) surface. However, the mechanisms involved remain unknown. By treating human umbilical vein endothelial cells (HUVECs) with VEGF analogs, we show that activation of VEGFR-1 or VEGFR-2 or both induced a rapid and transient translocation of endothelial P-selectin and neutrophil adhesion to activated ECs. The effects mediated by VEGF-A(165) and VEGF-A(121) (VEGFR-1/VEGFR-2 agonists) were blocked by a selective VEGFR-2 inhibitor, SU1498. VEGF-A(165) was twice as potent as VEGF-A(121), which can be explained by the binding capacity of VEGF-A(165) to its coreceptor neuropilin-1 (NRP-1). Indeed, treatment with NRP-1 antagonist (GST-Ex7) reduced the effect of VEGF-A(165) to the levels observed upon stimulation with VEGF-A(121). Finally, the use of selective PAF receptor antagonists reduced VEGF-A(165)-mediated P-selectin translocation. Together, these data show that maximal P-selectin translocation and subsequent neutrophil adhesion was mediated by VEGF-A(165) on the activation of VEGFR-2/NRP-1 complex and required PAF synthesis.
Asunto(s)
Endotelio Vascular/citología , Selectina-P/metabolismo , Factor de Activación Plaquetaria/fisiología , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Humanos , Neuropilina-1/metabolismo , Neutrófilos/citología , Factor de Activación Plaquetaria/biosíntesis , Transporte de Proteínas , Receptores de Factores de Crecimiento Endotelial Vascular/fisiología , Venas Umbilicales/citología , Factor A de Crecimiento Endotelial Vascular/análogos & derivados , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) are two inflammatory mediators capable of promoting endothelial cell (EC) migration and angiogenesis. As VEGF inflammatory effect is mediated by the synthesis of endothelial platelet-activating factor (PAF) which is also contributing to VEGF chemotactic activity, we wanted to assess if S1P can trigger PAF synthesis in EC and if S1P-induced migration is PAF-dependent. Treatment of bovine aortic EC (BAEC) with S1P (10(-10)-10(-6) M) increased dose- and time-dependently the synthesis of PAF by up to 3.3-fold above the basal level, with a maximal amount of PAF detected at 20 min post-stimulation. This biological response was attenuated by inhibiting p38 mitogen-activated protein kinase (MAPK), cytosolic or secreted phospholipase A(2) (cPLA(2), sPLA(2)) activity, suggesting that p38 MAPK activation by S1P promotes the conversion of membrane phospholipids into PAF through the combined activation of cPLA(2) and sPLA(2). Interestingly, pretreatment of BAEC with extracellular PAF receptor antagonists (BN52021, 10(-5) M and CV3988, 10(-6) M) reduced by up to 42% the cellular migration induced by S1P (10(-6) M). These data demonstrate the capacity of S1P to induce PAF synthesis, which contributes in part to S1P chemotactic activity.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Lisofosfolípidos/farmacología , Factor de Activación Plaquetaria/biosíntesis , Proteínas Serina-Treonina Quinasas , Esfingosina/farmacología , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Bovinos , Células Cultivadas , Células Endoteliales/citología , Activación Enzimática/efectos de los fármacos , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/metabolismo , Fosforilación/efectos de los fármacos , Factor de Activación Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Esfingosina/análogos & derivados , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
1. The mechanism(s) by which vascular endothelial growth factor (VEGF) induces endothelial nitric oxide synthase (eNOS) activation remain(s) unclear up to a certain extent. Therefore, we sought to evaluate the contribution of numerous pathways in VEGF-induced nitric oxide (NO) synthesis by measuring cGMP production. In addition, as VEGF induces the synthesis of NO and platelet-activating factor (PAF), we wanted to assess if the induction of PAF and NO is contributing to the synthesis of each other. 2. Herein, we show that a treatment of endothelial cells with a phospholipase C (PLC) inhibitor (U73122), a calmodulin antagonist (W-7) or with intracellular calcium chelators (EGTA/AM, BAPTA/AM) prevented VEGF-mediated eNOS Ser(1177)-phosphorylation and NO synthesis measured by cGMP production. 3. Pretreatment with phosphatidylinositol 3-kinase (PI3K) (Wortmannin, LY294002) or protein kinase C (PKC) (GF109203X, Ro318220) inhibitors attenuated eNOS Ser(1177)-phosphorylation mediated by VEGF, but did not alter immediate (0-10 min) cGMP synthesis induced by VEGF, but abrogated by up to 84% the delayed (10-30 min) cGMP synthesis. 4. Pretreatment with PAF synthesis inhibitors or with PAF receptor antagonists did not abrogate neither eNOS Ser(1177)-phosphorylation nor cGMP synthesis mediated by VEGF. 5. In conclusion, VEGF induces an immediate cGMP synthesis through the PLC-Ca2+/CaM pathway, and that the induction of delayed cGMP synthesis implies Akt and PKC activity.
Asunto(s)
Diterpenos , Ácido Egtácico/análogos & derivados , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Linfocinas/farmacología , Óxido Nítrico/metabolismo , Proteínas Serina-Treonina Quinasas , Receptores Acoplados a Proteínas G , 1-Metil-3-Isobutilxantina/farmacología , Androstadienos/farmacología , Animales , Calcimicina/farmacología , Calcio/antagonistas & inhibidores , Bovinos , Células Cultivadas , Quelantes/farmacología , Cromonas/farmacología , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Ácido Egtácico/farmacología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Ginkgólidos , Indoles/farmacología , Ionóforos/farmacología , Lactonas/farmacología , Maleimidas/farmacología , Morfolinas/farmacología , NG-Nitroarginina Metil Éster/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Nitroprusiato/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Factor de Activación Plaquetaria/metabolismo , Factor de Activación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores de Superficie Celular/antagonistas & inhibidores , Serina/metabolismo , Transducción de Señal/fisiología , Factores de Tiempo , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , WortmaninaRESUMEN
Vascular endothelial growth factor (VEGF-A) is an inducer of endothelial cell (EC) proliferation, migration, and synthesis of inflammatory agents such as platelet-activating factor (PAF). Recently, neuropilin-1 (NRP-1) has been described as a coreceptor of KDR which potentiates VEGF-A activity. However, the role of NRP-1 in numerous VEGF-A activities remains unclear. To assess the contribution of NRP-1 to VEGF-A mediated EC proliferation, migration, and PAF synthesis, we used porcine aortic EC (PAEC) recombinantly expressing Flt-1, NRP-1, KDR or KDR and NRP-1. Cells were stimulated with VEGF-A, which binds to Flt-1, KDR and NRP-1, and VEGF-C, which binds to KDR only. VEGF-A was 12.4-fold more potent than VEGF-C in inducing KDR phosphorylation in PAEC-KDR. VEGF-A and VEGF-C showed similar potency to mediate PAEC-KDR proliferation, migration, and PAF synthesis. On PAEC-KDR/NRP-1, VEGF-A was 28.6-fold more potent than VEGF-C in inducing KDR phosphorylation and PAEC-KDR/NRP-1 proliferation (1.3-fold), migration (1.7-fold), and PAF synthesis (4.6-fold). These results suggest that cooperative binding of VEGF-A to KDR and NRP-1 enhances KDR phosphorylation and its biological activities. Similar results were obtained with bovine aortic EC that endogenously express both KDR and NRP-1 receptors. In contrast, stimulation of PAEC-Flt-1 and PAEC-NRP-1 with VEGF-A or VEGF-C did not induce proliferation, migration, or PAF synthesis. In conclusion, the presence of NRP-1 on EC preferentially increases KDR activation by VEGF-A as well as KDR-mediated biological activities, and may elicit novel intracellular events. On the other hand, VEGF-A and VEGF-C have equipotent biological activities on EC in absence of NRP-1.