RESUMO
Endothelial cell-specific chemotaxis receptor (ECSCR) is a cell surface protein expressed by blood endothelial cells with roles in endothelial cell migration and signal transduction. We investigated the function of ecscr in the development of the zebrafish vasculature. Zebrafish ecscr is expressed in angioblasts and in axial vessels during angioblast migration and vasculogenesis. Morpholino-directed ecscr knockdown resulted in defective angioblast migration in the posterior lateral plate mesoderm, a process known to depend on vascular endothelial-derived growth factor (VEGF). In cultured cells, transfected ECSCR localized to actin-rich membrane protrusions, colocalizing with kinase insert domain protein receptor (KDR)/VEGF receptor 2 in these regions. ECSCR-silenced cells show reduced VEGF-induced phosphorylation of KDR but not of FMS-like tyrosine kinase 1 (FLT1)/VEGF receptor 1. Finally, chemical inhibition of VEGF receptor activity in zebrafish resulted in angioblast deficiencies that partially overlap with those seen in ecscr morphants. We propose that ecscr promotes migration of zebrafish angioblasts by enhancing endothelial kdr sensitivity to VEGF.
Assuntos
Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Quimiotaxia/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Sequência de Bases , Movimento Celular , Proliferação de Células , Células Cultivadas , Primers do DNA/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Transfecção , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The Nogo-B receptor (NgBR) is a recently identified receptor for the N terminus of reticulon 4B/Nogo-B. Other than its role in binding Nogo-B, little is known about the biology of NgBR. To elucidate a basic cellular role for NgBR, we performed a yeast two-hybrid screen for interacting proteins, using the C-terminal domain as bait, and identified Niemann-Pick type C2 protein (NPC2) as an NgBR-interacting protein. NPC2 protein levels are increased in the presence of NgBR, and NgBR enhances NPC2 protein stability. NgBR localizes primarily to the endoplasmic reticulum (ER) and regulates the stability of nascent NPC2. RNAi-mediated disruption of NgBR or genetic deficiency in NgBR lead to a decrease in NPC2 levels, increased intracellular cholesterol accumulation, and a loss of sterol sensing, all hallmarks of an NPC2 mutation. These data identify NgBR as an NPC2-interacting protein and provide evidence of a role for NgBR in intracellular cholesterol trafficking.
Assuntos
Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Glicoproteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Células CHO , Linhagem Celular , LDL-Colesterol/metabolismo , Cricetinae , Cricetulus , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Humanos , RNA Interferente Pequeno/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Transporte VesicularRESUMO
Nogo isoforms (Nogo-A and -B) have been implicated in regulating neural and cardiovascular functions, such as cell spreading and chemotaxis. Unlike the loop domain (Nogo-66) found in all Nogo isoforms that can interact with a neural-specific Nogo-66 receptor, the receptor for the amino terminus of Nogo-B that mediates vascular function is unknown. Here, we identify a previously uncharacterized Nogo-B receptor specific for the amino terminus of Nogo-B and show that Nogo-B receptor localizes with the ligand Nogo-B during VEGF and wound healing angiogenesis in vivo, mediates chemotaxis in a heterologous expression system and chemotaxis, and 3D tube formation in native endothelial cells. Thus, identification of this receptor may lead to the discovery of agonists or antagonists of this pathway to regulate vascular remodeling and angiogenesis.
Assuntos
Forma Celular , Quimiotaxia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteínas da Mielina/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas da Mielina/genética , Proteínas da Mielina/farmacologia , Proteínas Nogo , Ligação Proteica , Isoformas de Proteínas/metabolismo , Receptores de Superfície Celular/químicaRESUMO
Although Nogo-A has been identified in the central nervous system as an inhibitor of axonal regeneration, the peripheral roles of Nogo isoforms remain virtually unknown. Here, using a proteomic analysis to identify proteins enriched in caveolae and/or lipid rafts (CEM/LR), we show that Nogo-B is highly expressed in cultured endothelial and smooth muscle cells, as well as in intact blood vessels. The N terminus of Nogo-B promotes the migration of endothelial cells but inhibits the migration of vascular smooth muscle (VSM) cells, processes necessary for vascular remodeling. Vascular injury in Nogo-A/B-deficient mice promotes exaggerated neointimal proliferation, and adenoviral-mediated gene transfer of Nogo-B rescues the abnormal vascular expansion in those knockout mice. Our discovery that Nogo-B is a regulator of vascular homeostasis and remodeling broadens the functional scope of this family of proteins.
