RESUMEN
The central pathway for oxygen-dependent control of red cell mass is the prolyl hydroxylase domain protein (PHD):hypoxia inducible factor (HIF) pathway. PHD site specifically prolyl hydroxylates the transcription factor HIF-α, thereby targeting the latter for degradation. Under hypoxia, this modification is attenuated, allowing stabilized HIF-α to activate target genes, including that for erythropoietin (EPO). Studies employing genetically modified mice point to Hif-2α, one of two main Hif-α isoforms, as being the critical regulator of Epo in the adult mouse. More recently, erythrocytosis patients with heterozygous point mutations in the HIF2A gene have been identified; whether these mutations were polymorphisms unrelated to the phenotype could not be ruled out. In the present report, we characterize a mouse line bearing a G536W missense mutation in the Hif2a gene that corresponds to the first such human mutation identified (G537W). We obtained mice bearing both heterozygous and homozygous mutations at this locus. We find that these mice display, in a mutation dose-dependent manner, erythrocytosis and pulmonary hypertension with a high degree of penetrance. These findings firmly establish missense mutations in HIF-2α as a cause of erythrocytosis, highlight the importance of this HIF-α isoform in erythropoiesis, and point to physiologic consequences of HIF-2α dysregulation.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Hipertensión Pulmonar/genética , Mutación Missense , Policitemia/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Análisis de los Gases de la Sangre , Células Cultivadas , Modelos Animales de Enfermedad , Endotelina-1/genética , Endotelina-1/metabolismo , Eritropoyesis , Eritropoyetina/sangre , Eritropoyetina/genética , Expresión Génica , Técnicas de Sustitución del Gen , Estudios de Asociación Genética , Humanos , Hipertensión Pulmonar/sangre , Hipertensión Pulmonar/fisiopatología , Hipertrofia Ventricular Derecha/sangre , Hipertrofia Ventricular Derecha/genética , Hipertrofia Ventricular Derecha/fisiopatología , Riñón/metabolismo , Pulmón/metabolismo , Pulmón/patología , Pulmón/fisiopatología , Ratones , Ratones Endogámicos C57BL , Mutagénesis , Policitemia/sangre , Policitemia/fisiopatología , Proteínas Proto-Oncogénicas c-sis/genética , Proteínas Proto-Oncogénicas c-sis/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Frecuencia Respiratoria , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/sangreRESUMEN
The central pathway for controlling red cell mass is the PHD (prolyl hydroxylase domain protein):hypoxia-inducible factor (HIF) pathway. HIF, which is negatively regulated by PHD, activates numerous genes, including ones involved in erythropoiesis, such as the ERYTHROPOIETIN (EPO) gene. Recent studies have implicated PHD2 as the key PHD isoform regulating red cell mass. Studies of humans have identified erythrocytosis-associated, heterozygous point mutations in the PHD2 gene. A key question concerns the mechanism by which human mutations lead to phenotypes. In the present report, we generated and characterized a mouse line in which a P294R knock-in mutation has been introduced into the mouse Phd2 locus to model the first reported human PHD2 mutation (P317R). Phd2(P294R/+) mice display a degree of erythrocytosis equivalent to that seen in Phd2(+/-) mice. The Phd2(P294R/+)-associated erythrocytosis is reversed in a Hif2a(+/-), but not a Hif1a(+/-) background. Additional studies using various conditional knock-outs of Phd2 reveal that erythrocytosis can be induced by homozygous and heterozygous knock-out of Phd2 in renal cortical interstitial cells using a Pax3-Cre transgene or by homozygous knock-out of Phd2 in hematopoietic progenitors driven by a Vav1-Cre transgene. These studies formally prove that a missense mutation in PHD2 is the cause of the erythrocytosis, show that this occurs through haploinsufficiency, and point to multifactorial control of red cell mass by PHD2.
Asunto(s)
Haploinsuficiencia , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Mutación Missense , Policitemia/metabolismo , Sustitución de Aminoácidos , Animales , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Ratones , Ratones Transgénicos , Policitemia/genética , Policitemia/patologíaRESUMEN
The original version of this Article contained an error in the spelling of the author Brett L. Ecker, which was incorrectly given as Brett Ecker. This has now been corrected in both the PDF and HTML versions of the Article.
RESUMEN
Prolyl hydroxylase domain protein 2 (PHD2) is a well-known master oxygen sensor. However, the role of PHD2 in tumor initiation remains controversial. We find that during the transition of human nevi to melanoma, the expression of PHD2 protein is significantly decreased and lower expression PHD2 in melanoma is associated with worse clinical outcome. Knockdown of PHD2 leads to elevated Akt phosphorylation in human melanocytes. Mice with conditional melanocyte-specific expression of Phd2lox/lox (Tyr::CreER;Phd2lox/lox) fail to develop pigmented lesions. However, deletion of Phd2 in combination with expression of BRafV600E in melanocytes (Tyr::CreER;Phd2lox/lox;BRafCA) leads to the development of melanoma with 100% penetrance and frequent lymph node metastasis. Analysis of tumor tissues using reverse phase protein arrays demonstrates that Phd2 deletion activates the AKT-mTOR-S6 signaling axis in the recovered tumors. These data indicate that PHD2 is capable of suppressing tumor initiation largely mediated through inhibiting of the Akt-mTOR signaling pathway in the melanocyte lineage.