RESUMO
Placental development initially occurs in a low-oxygen (O2) or hypoxic environment. In this report we show that two hypoxia-inducible factors (HIFs), HIF1alpha and HIF2alpha, are essential for determining murine placental cell fates. HIF is a heterodimer composed of HIFalpha and HIFbeta (ARNT) subunits. Placentas from Arnt-/- and Hif1alpha-/- Hif2alpha-/- embryos exhibit defective placental vascularization and aberrant cell fate adoption. HIF regulation of Mash2 promotes spongiotrophoblast differentiation, a prerequisite for trophoblast giant cell differentiation. In the absence of Arnt or Hifalpha, trophoblast stem cells fail to generate these cell types and become labyrinthine trophoblasts instead. Therefore, HIF mediates placental morphogenesis, angiogenesis, and cell fate decisions, demonstrating that O2 tension is a critical regulator of trophoblast lineage determination. This novel genetic approach provides new insights into the role of O2 tension in the development of life-threatening pregnancy-related diseases such as preeclampsia.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismo , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/deficiência , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Feminino , Células Gigantes/citologia , Células Gigantes/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Knockout , Fenótipo , Placenta/anormalidades , Placenta/irrigação sanguínea , Placenta/citologia , Placenta/metabolismoRESUMO
Inactivating mutations within the von Hippel-Lindau (VHL) tumor suppressor gene predispose patients to develop a variety of highly vascularized tumors. pVHL targets alpha subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF), a critical regulator of energy metabolism, angiogenesis, hematopoiesis, and oxygen (O(2)) delivery, for ubiquitin-mediated degradation in an O(2)-dependent manner. To investigate the role of Vhl in cellular proliferation and tumorigenesis, we utilized mouse embryonic fibroblasts (MEFs), a common tool for analyzing cell cycle regulation, and generated Vhl(-)(/)(-) MEF-derived fibrosarcomas. Surprisingly, growth of both Vhl(-)(/)(-) MEFs and fibrosarcomas was impaired, although tumor vascularity was increased. Decreased proliferation of Vhl(-)(/)(-) MEFs was correlated with an overexpression of cyclin kinase inhibitors (CKIs) p21 and p27. The transcription of p21 and p27 is inhibited by c-Myc; therefore, the induction of CKIs was attributed to the ability of HIF to antagonize c-Myc activity. Indeed, p21 mRNA levels were elevated under normoxia in Vhl(-)(/)(-) MEFs, while c-Myc transcriptional activity was markedly reduced. Gene silencing of HIF-1alpha by small interfering RNA reduced p21 and p27 protein and mRNA levels in Vhl(-)(/)(-) MEFs. The induction of p21 and p27, mediated by constitutive activation of the HIF pathway, provides a mechanism for the decreased proliferation rates of Vhl(-)(/)(-) MEFs and fibrosarcomas. These results demonstrate that a loss of pVHL can induce growth arrest in certain cells types, which suggests that additional genetic mutations are necessary for VHL-associated tumorigenesis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fibrossarcoma/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Apoptose , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Embrião de Mamíferos/citologia , Feminino , Fibroblastos/metabolismo , Fibrossarcoma/genética , Fibrossarcoma/patologia , Inativação Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia , Camundongos , Camundongos Mutantes , Mutação , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
Inactivation of the von Hippel-Lindau (VHL) gene is associated with the development of highly vascularized tumors. pVHL targets the alpha subunits of hypoxia inducible factor (HIF) for ubiquitin-mediated degradation in an oxygen-dependent manner. Although pVHL-deficient tumor cell lines demonstrate constitutive stabilization and activation of HIF, it has yet to be shown that loss of murine Vhl alone is sufficient to dysregulate HIF. We utilized a genetic approach to demonstrate that loss of Vhl is sufficient not only to stabilize HIF-alpha subunits under normoxia, but also fully activate HIF-mediated responses. These studies have implications for the hierarchy of signaling events leading to HIF stabilization, nuclear translocation, and target gene expression. We further demonstrate that loss of murine Vhl does not promote teratocarcinoma growth, indicating that other genetic changes must occur to facilitate Vhl-mediated tumorigenesis.
