RESUMO
Sorting nexins 1 (Snx1) and 2 (Snx2) are homologues of the yeast gene VPS5 that is required for proper endosome-to-Golgi trafficking. The prevailing thought is that Vps5p is a component of a retrograde trafficking complex called the retromer. Genetic and biochemical evidence suggest mammals may have similar complexes, but their biological role is unknown. Furthermore, if SNX1 and SNX2 belong to such complexes, it is not known whether they act together or separately. Herein, we show that mice lacking SNX1 or SNX2 are viable and fertile, whereas embryos deficient in both proteins arrest at midgestation. These results demonstrate that SNX1 and SNX2 have a highly redundant and necessary function in the mouse. The phenotype of Snx1(-/-);Snx2(-/-) embryos is very similar to that of embryos lacking another retromer homologue, Hbeta58. This finding suggests that SNX1/SNX2 and Hbeta58 function in the same genetic pathway, providing additional evidence for the existence of mammalian complexes that are structurally similar to the yeast retromer. Furthermore, the viability of Snx1(-/-) and Snx2(-/-) mice demonstrates that it is not necessary for SNX1 and SNX2 to act together. Electron microscopy indicates morphological alterations of apical intracellular compartments in the Snx1(-/-);Snx2(-/-) yolk-sac visceral endoderm, suggesting SNX1 and SNX2 may be required for proper cellular trafficking. However, tetraploid aggregation experiments suggest that yolk sac defects cannot fully account for Snx1(-/-); Snx2(-/-) embryonic lethality. Furthermore, endocytosis of transferrin and low-density lipoprotein is unaffected in mutant primary embryonic fibroblasts, indicating that SNX1 and SNX2 are not essential for endocytosis in all cells. Although the two proteins demonstrate functional redundancy, Snx1(+/-);Snx2(-/-) mice display abnormalities not observed in Snx1(-/-);Snx2(+/-) mice, revealing that SNX1 and SNX2, or their genetic regulation, are not equivalent. Significantly, these studies represent the first mutations in the mammalian sorting nexin gene family and indicate that sorting nexins perform essential functions in mammals.
Assuntos
Proteínas de Transporte/fisiologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário e Fetal , Proteínas de Transporte Vesicular , Animais , Animais Recém-Nascidos , Proteínas de Transporte/genética , Embrião de Mamíferos/anatomia & histologia , Endocitose/fisiologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Retardo do Crescimento Fetal/genética , Viabilidade Fetal , Fibroblastos/citologia , Fibroblastos/metabolismo , Marcação de Genes , Substâncias Macromoleculares , Masculino , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Morfogênese , Fenótipo , Gravidez , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/fisiologia , Saco Vitelino/metabolismo , Saco Vitelino/ultraestruturaAssuntos
Aborto Induzido , Direitos Civis , Regulamentação Governamental , Jurisprudência , Padrões de Referência , Controle Social Formal , Decisões da Suprema Corte , Adolescente , Desenvolvimento Embrionário e Fetal , Financiamento Governamental , Liberdade , Humanos , Consentimento Livre e Esclarecido , Função Jurisdicional , Menores de Idade , Consentimento dos Pais , Notificação aos Pais , Pais , Autonomia Pessoal , Gravidez , Gestantes , Privacidade , Cônjuges , Governo Estadual , Consentimento do Representante Legal , Estados UnidosRESUMO
The interaction between HIF-1alpha, Mdm2, and p53 proteins during hypoxia has received recent attention. Here, we investigated the consequences of interaction between HIF-1alpha and Mdm2 under hypoxic conditions. Endogenous HIF-1alpha and Mdm2 proteins were co-immunoprecipitated from lysates of hypoxic HCT116 p53WT and p53(-/-) cells, suggesting that association of these two proteins is a p53-independent event. The cellular Mdm2 protein content was not significantly altered in hypoxic tumor cells. Overexpression of Mdm2 resulted in an increase in HIF-1alpha protein content in hypoxic cells and increased hypoxia-induced vascular endothelial growth factor (VEGF) transcriptional activation. These results point toward a novel and p53-independent function of Mdm2 to promote tumor cell adaptations to hypoxia by interacting with and promoting HIF-1 activation.