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1.
Thromb Haemost ; 118(1): 132-142, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29304533

RESUMO

Sphingosine-1-phosphate (S1P) is a potent lipid mediator released from activated platelets by an adenosine triphosphate (ATP)-dependent export mechanism. A candidate transport protein is the multidrug resistance protein 4 (MRP4/ABCC4), an ATP-dependent transporter highly expressed in platelets. Furthermore, several statins are known to affect platelet functions and exhibit antithrombotic properties. This study determines the involvement of MRP4 in the transport of S1P and a possible interference by statins. Transport studies in membrane vesicles of Sf9 cells containing recombinant human MRP4 revealed that MRP4 mediates ATP-dependent transport of fluorescein- and tritium-labelled S1P. Also, ATP-dependent S1P transport in platelet membrane vesicles containing endogenous MRP4 was inhibited by the MRP inhibitor MK571 and the MRP4-selective compound Ceefourin-1. Confocal microscopy using fluorescein-labelled S1P as well as boron-dipyrromethene (BODIPY)-labelled sphingosine indicated association of S1P and MRP4 in human platelets. In MRP4-deficient mice, agonist-induced S1P secretion was reduced compared with matched wild-type C57Bl/6 mice and platelet S1P concentrations were lower. Fluvastatin and rosuvastatin interfered with MRP4 function inhibiting ATP-dependent cGMP (cyclic guanosine monophosphate) uptake into MRP4-containing vesicles, inhibited MRP4-mediated S1P transport in vitro and significantly attenuated endogenous S1P release from agonist-activated platelet ex vivo. These data suggest that release of S1P from platelets depends on MRP4 and statins can interfere with this transport process. Potentially, this may be relevant for the pleiotropic anti-inflammatory effects of statins and their effect on modulating atherothrombosis.


Assuntos
Plaquetas/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lisofosfolipídeos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Esfingosina/análogos & derivados , Animais , Transporte Biológico , Compostos de Boro , Cromatografia Líquida , Fluvastatina/farmacologia , Voluntários Saudáveis , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Ativação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária , Proteínas Recombinantes/metabolismo , Rosuvastatina Cálcica/farmacologia , Células Sf9 , Esfingosina/metabolismo , Espectrometria de Massas em Tandem
2.
Thromb Haemost ; 117(1): 105-115, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-27761583

RESUMO

The multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit ß3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knock-down of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leukaemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.


Assuntos
Complexo 3 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Plaquetas/metabolismo , Membrana Celular/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Leucemia Megacarioblástica Aguda/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Complexo 3 de Proteínas Adaptadoras/química , Complexo 3 de Proteínas Adaptadoras/genética , Subunidades beta do Complexo de Proteínas Adaptadoras/química , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Animais , Plaquetas/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Proteína 4 Homóloga a Disks-Large/química , Proteína 4 Homóloga a Disks-Large/genética , Cães , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Leucemia Megacarioblástica Aguda/genética , Leucemia Megacarioblástica Aguda/patologia , Macrolídeos/farmacologia , Células Madin Darby de Rim Canino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Interferência de RNA , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Transfecção
3.
Pharmacogenet Genomics ; 24(10): 522-6, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25121519

RESUMO

Multidrug resistance protein 4 (MRP4/ABCC4) has been established as an independent regulator of cyclic AMP (cAMP) levels particularly in vascular smooth muscle cells and in hematopoietic cells. Here, we assessed whether cAMP in turn regulates MRP4. A significant upregulation of MRP4 mRNA and protein by long-term treatment with cAMP-enhancing agents was observed in HeLa cells, smooth muscle cells, and megakaryoblastic leukemia M07e cells. This upregulation was not affected by inhibition of protein kinase A, but could be reverted by inhibitors and siRNA of an alternative cAMP-signaling route involving exchange proteins activated by cyclic AMP (EPAC) and mitogen-activated protein kinases. A selective EPAC activator could equally induce MRP4. The transcriptional regulation was confirmed in a luciferase reporter gene assay using a vector containing a 1494-bp fragment of the promoter region of the MRP4/ABCC4 gene. Our results suggest that enhanced cAMP levels upregulate MRP4 expression, which can result in increased cAMP efflux.


Assuntos
AMP Cíclico/análogos & derivados , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Células Musculares/metabolismo , Músculo Liso/citologia , Transdução de Sinais , Células Cultivadas , AMP Cíclico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Músculo Liso/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima
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