RESUMEN
BACKGROUND: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique proteolytic activation mechanism. PARs regulate a broad range of cellular functions and are involved in the pathogenesis of inflammatory disorders. Moreover, PAR1 and PAR2 activation in the endothelium shifts it toward a prothrombotic condition. OBJECTIVES: To assess the relevance of intracellular reactive oxygen species (ROS) in the signaling events underlying tissue factor (TF) expression elicited by PAR1 and PAR2 occupancy in endothelial cells, and to investigate their source. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed to specific PAR1 and PAR2 agonist peptides. TF expression was determined by real-time reverse transcription polymerase chain reaction analysis and measurement of procoagulant activity. ROS generation was determined by a fluorometric assay after cell loading with 2'-7'-dichlorofluorescein diacetate. RESULTS: ROS generated by the mitochondrial chain, mostly from complex III, provide a pathway through which PAR1 and PAR2 occupancy induces TF. Other sources of ROS do not participate in TF induction. Activation of both ERK1/2 and p38 MAPK is critical for mitochondrial ROS generation. In addition to these pathways shared by the two PARs, mechanisms downstream from PAR1 and PAR2 activation, different for the two receptors, also induced TF. A module that sensitively regulates PAR1 signaling and ultimately involves NF-kappaB activation has been identified. CONCLUSIONS: Our data identify ROS originating in mitochondria as key mediators of the signaling pathways triggered by PAR1 and PAR2 engagement in endothelial cells and show that downstream from receptor activation occur cascades that are mechanistically coupled to procoagulant activity.
Asunto(s)
Células Endoteliales/metabolismo , Mitocondrias/metabolismo , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Tromboplastina/genética , Coagulación Sanguínea , Células Cultivadas , Células Endoteliales/ultraestructura , Endotelio Vascular/citología , Humanos , Especies Reactivas de Oxígeno , Transducción de Señal/fisiología , Activación TranscripcionalRESUMEN
BACKGROUND: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique mechanism of proteolytic activation. PARs regulate a broad range of cellular functions and are active in the pathogenesis of disorders characterized by chronic inflammation or activation of the coagulation cascade. Signaling through PAR1 and PAR2 shifts the endothelium towards a prothrombotic phenotype, thereby exacerbating the initial pathophysiologic condition. OBJECTIVES: This study aimed to analyze the localization of PARs in the cell membrane and how their compartmentalization affects tissue factor (TF) in human endothelial cells. METHODS: TF expression was determined by quantitative real-time polymerase chain reaction analysis and by activity assays. The interaction of PARs with caveolin was investigated through: (i) caveolin-1 gene knockdown performed by transfection with specific small interfering RNA (siRNA); (ii) caveolin-enriched membrane microdomain disruption; and (iii) coimmunoprecipitation assay. RESULTS: We have shown that PAR1, but not PAR2, is present in endothelial caveolin-enriched membrane microdomains, where it is bound to caveolin-1, and that these structures must be intact if PAR1-induced signaling is to increase TF activity. Cholesterol depletion of endothelial cells by cholesterol-sequestering agents caused the PAR1 to relocate to high-density membranes, and impaired the induction of TF (P < 0.01) without affecting the PAR2-mediated procoagulant effect. In addition, siRNA directed against caveolin-1 inhibited TF activation by PAR1 (P < 0.01 and P < 0.01, respectively). CONCLUSIONS: PAR1 localization in the caveolin-enriched membrane microdomain, bound to caveolin-1, represents a crucial requirement for TF induction in endothelial cells.
Asunto(s)
Caveolina 1/metabolismo , Células Endoteliales/metabolismo , Microdominios de Membrana/metabolismo , Receptor PAR-1/metabolismo , Transducción de Señal , Tromboplastina/biosíntesis , Caveolina 1/genética , Células Cultivadas , Colesterol/deficiencia , Colesterol/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Humanos , Microdominios de Membrana/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Nistatina/farmacología , Oligopéptidos/farmacología , Fosforilación , Transporte de Proteínas , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/metabolismo , Receptor PAR-1/agonistas , Receptor PAR-2/metabolismo , Transducción de Señal/efectos de los fármacos , Tromboplastina/genética , TransfecciónRESUMEN
A series of novel C2-exo unsaturated pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) has been synthesised via a versatile pro-C2 ketone precursor. C2-exo-unsaturation enhances both DNA-binding reactivity and in vitro cytotoxic potency.
Asunto(s)
Antibióticos Antineoplásicos/química , Benzodiazepinas/química , ADN/metabolismo , Pirroles/química , Animales , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacología , Benzodiazepinas/metabolismo , Benzodiazepinas/farmacología , Benzodiazepinonas/química , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Estructura Molecular , Neoplasias Ováricas , Pirroles/metabolismo , Pirroles/farmacología , Células Tumorales CultivadasRESUMEN
A series of novel C2,C3-endo unsaturated pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) has been synthesised via cleavage of the N10-Alloc protecting group from appropriate precursors. Biophysical and biological evaluations show that the presence of C2/C3-endo unsaturation in the PBD C-ring enhances both DNA-binding reactivity and in vitro cytotoxic potency.
