RESUMEN
Sphingosine-1-phosphate (S1P) participates in inflammation; however, its role in leukocyte rolling is still unclear. Here we use intravital microscopy in inflamed mouse cremaster muscle venules and human endothelial cells to show that S1P contributes to P-selectin-dependent leukocyte rolling through endothelial S1P receptor 3 (S1P3) and Gαq, PLCß and Ca(2+). Intra-arterial S1P administration increases leukocyte rolling, while S1P3 deficiency or inhibition dramatically reduces it. Mast cells involved in triggering rolling also release S1P that mobilizes P-selectin through S1P3. Histamine and epinephrine require S1P3 for full-scale effect accomplishing it by stimulating sphingosine kinase 1 (Sphk1). In a counter-regulatory manner, S1P1 inhibits cAMP-stimulated Sphk1 and blocks rolling as observed in endothelial-specific S1P1(-/-) mice. In agreement with a dominant pro-rolling effect of S1P3, FTY720 inhibits rolling in control and S1P1(-/-) but not in S1P3(-/-) mice. Our findings identify S1P as a direct and indirect contributor to leukocyte rolling and characterize the receptors mediating its action.
Asunto(s)
Endotelio Vascular/metabolismo , Rodamiento de Leucocito/genética , Selectina-P/metabolismo , Receptores de Lisoesfingolípidos/genética , Animales , Calcio/metabolismo , Epinefrina/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Histamina/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunohistoquímica , Lisofosfolípidos/metabolismo , Masculino , Mastocitos/metabolismo , Ratones , Ratones Noqueados , Músculo Esquelético/irrigación sanguínea , Fosfolipasa C beta/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato , VénulasRESUMEN
Embryonic development depends on complex and precisely orchestrated signaling pathways including specific reduction/oxidation cascades. Oxidoreductases of the thioredoxin family are key players conveying redox signals through reversible posttranslational modifications of protein thiols. The importance of this protein family during embryogenesis has recently been exemplified for glutaredoxin 2, a vertebrate-specific glutathione-disulfide oxidoreductase with a critical role for embryonic brain development. Here, we discovered an essential function of glutaredoxin 2 during vascular development. Confocal microscopy and time-lapse studies based on two-photon microscopy revealed that morpholino-based knockdown of glutaredoxin 2 in zebrafish, a model organism to study vertebrate embryogenesis, resulted in a delayed and disordered blood vessel network. We were able to show that formation of a functional vascular system requires glutaredoxin 2-dependent reversible S-glutathionylation of the NAD(+)-dependent protein deacetylase sirtuin 1. Using mass spectrometry, we identified a cysteine residue in the conserved catalytic region of sirtuin 1 as target for glutaredoxin 2-specific deglutathionylation. Thereby, glutaredoxin 2-mediated redox regulation controls enzymatic activity of sirtuin 1, a mechanism we found to be conserved between zebrafish and humans. These results link S-glutathionylation to vertebrate development and successful embryonic angiogenesis.
Asunto(s)
Sistema Cardiovascular/embriología , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Neovascularización Fisiológica/fisiología , Transducción de Señal/fisiología , Sirtuina 1/metabolismo , Animales , Western Blotting , Cartilla de ADN/genética , Técnicas de Silenciamiento del Gen , Glutarredoxinas/genética , Células HeLa , Humanos , Espectrometría de Masas , Microscopía Confocal , Oxidación-Reducción , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genética , Imagen de Lapso de Tiempo , Pez CebraRESUMEN
Motexafin gadolinium (MGd) sensitizes malignant cells to ionizing radiation, although the underlying mechanisms for uptake and sensitization are both unclear. Here we show that MGd is endocytosed by the clathrin-dependent pathway with ensuing lysosomal membrane permeabilization, most likely via formation of reactive oxygen species involving redox-active metabolites, such as ascorbate. We propose that subsequent apoptosis is a synergistic effect of irradiation and high MGd concentrations in malignant cells due to their pronounced endocytic activity. The results provide novel insights into the mode of action of this promising anti-cancer drug, which is currently under clinical trials.