RESUMEN
Placental growth factor (PlGF) plays an important role in various pathological conditions and diseases such as inflammation, cancer, atherosclerosis and sickle cell disease (SCD). Abnormally high PlGF levels in SCD patients are associated with increased inflammation and pulmonary hypertension (PHT) and reactive airway disease; however, the transcriptional and post-transcriptional mechanisms regulating PlGF expression are not well defined. Herein, we show that treatment of human erythroid cells and colony forming units with erythropoietin (EPO) increased PlGF expression. Our studies showed EPO-mediated activation of HIF-1α led to subsequent binding of HIF-1α to hypoxia response elements (HREs) within the PlGF promoter, as demonstrated by luciferase transcription reporter assays and ChIP analysis of the endogenous gene. Additionally, we showed miR-214 post-transcriptionally regulated the expression of PlGF as demonstrated by luciferase reporter assays using wild-type (wt) and mutant PlGF-3'-UTR constructs. Furthermore, synthesis of miR-214, located in an intron of DNM3 (dynamin 3), was transcriptionally regulated by transcription factors, peroxisome proliferator-activated receptor-α (PPARα) and hypoxia-inducible factor-1α (HIF-1α). These results were corroborated in vivo wherein plasma from SCD patients and lung tissues from sickle mice showed an inverse correlation between PlGF and miR-214 levels. Finally, we observed that miR-214 expression could be induced by fenofibrate, a Food and Drug Administration (FDA) approved PPARα agonist, thus revealing a potential therapeutic approach for reduction in PlGF levels by increasing miR-214 transcription. This strategy has potential clinical implications for several pathological conditions including SCD.
Asunto(s)
Anemia de Células Falciformes/tratamiento farmacológico , Células Eritroides/efectos de los fármacos , Eritropoyetina/farmacología , Hematínicos/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/agonistas , MicroARNs/metabolismo , Proteínas Gestacionales/agonistas , Regiones no Traducidas 3'/efectos de los fármacos , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/metabolismo , Anemia de Células Falciformes/patología , Animales , Línea Celular , Células Cultivadas , Cruzamientos Genéticos , Células Eritroides/metabolismo , Células Eritroides/patología , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/patología , Eritropoyetina/uso terapéutico , Genes Reporteros/efectos de los fármacos , Hematínicos/uso terapéutico , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/sangre , Mutación , Factor de Crecimiento Placentario , Proteínas Gestacionales/antagonistas & inhibidores , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Endothelin-1, a potent vasoconstrictor, plays an important role in pulmonary hypertension (PH) in sickle cell disease (SCD). Our previous studies show that higher levels of placenta growth factor (PlGF), secreted by erythroid precursor cells, correlate with increased plasma levels of endothelin-1 (ET-1) and other functional markers of PH in SCD. PlGF-mediated ET-1 expression occurs via activation of hypoxia-inducible factor-1α (HIF-1α). However, relatively less is understood regarding how PlGF-mediated expression of HIF-1α and its downstream effector ET-1 are post-transcriptionally regulated. Herein, we show that PlGF treatment of endothelial cells resulted in reduced levels of miR-199a2, which targeted the 3'-UTR of HIF-1α mRNA and concomitantly led to augmented ET-1 expression. Plasma levels of miR-199a2 in SCD subjects were significantly lower with reciprocally high levels of plasma ET-1, unlike unaffected controls. This observation provided a molecular link between miR-199a2 and high levels of ET-1 in SCD. Furthermore, we show that miR-199a2 located in the DNM3os transcription unit was co-transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα). Binding of the latter to PPARα cis-elements in the promoter of DNM3os was demonstrated by promoter mutational analysis and ChIP. Additionally, we show that fenofibrate, a PPARα agonist, increased the expression of miR-199a2 and DNM3os; the former was responsible for reduced expression of HIF-1α and ET-1. In vivo studies of fenofibrate-fed Berkeley sickle mice resulted in increased levels of miR-199a2 and reduced levels of ET-1 in lung tissues. Our studies provide a potential therapeutic approach whereby fenofibrate-induced miR-199a2 expression can ameliorate PH by reduction of ET-1 levels.
Asunto(s)
Endotelina-1/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , MicroARNs/genética , PPAR alfa/fisiología , Transcripción Genética , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Dinamina III/genética , Endotelina-1/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones Endogámicos C57BL , MicroARNs/biosíntesis , Datos de Secuencia Molecular , Interferencia de ARNRESUMEN
The ß-hemoglobinopathies and thalassemias are serious genetic blood disorders affecting the ß-globin chain of hemoglobin A (α(2)ß(Α)(2)). Their clinical severity can be reduced by enhancing expression of fetal hemoglobin (γ-globin), producing HbF (α(2)γ(2,)). In studies reported here, γ-globin induction by 23 novel, structurally-unrelated compounds, which had been predicted through molecular modeling and in silico screening of a 13,000 chemical library, was evaluated in vitro in erythroid progenitors cultured from normal subjects and ß-thalassemia patients, and in vivo in transgenic mice or anemic baboons. Four predicted candidates were found to have high potency, with 4- to 8-fold induction of HbF. Two of these compounds have pharmacokinetic profiles favorable for clinical application. These studies thus effectively identified high potency γ-globin inducing candidate therapeutics and validated the utility of in silico molecular modeling.
Asunto(s)
Anemia/tratamiento farmacológico , Productos Biológicos/administración & dosificación , Diseño de Fármacos , Células Precursoras Eritroides/efectos de los fármacos , Hemoglobina Fetal/biosíntesis , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Talasemia beta/tratamiento farmacológico , gamma-Globinas/biosíntesis , Administración Oral , Anemia/genética , Anemia/metabolismo , Animales , Productos Biológicos/química , Productos Biológicos/uso terapéutico , Células Cultivadas , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/metabolismo , Hemoglobina Fetal/genética , Expresión Génica , Humanos , Inyecciones Intravenosas , Ratones , Ratones Transgénicos , Modelos Moleculares , Papio , Flebotomía , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Globinas beta/deficiencia , Globinas beta/genética , Talasemia beta/genética , Talasemia beta/metabolismo , gamma-Globinas/genéticaRESUMEN
Beta protein 1 (BP1), a human homeotic transcription factor, is expressed during hematopoeisis in the erythroid lineage. To determine the in vivo role of BP1 in erythropoiesis, we have undertaken two complementary approaches using enforced BP1 expression in both transgenic mice and embryonic stem (ES) cells. Despite repeated attempts, only one adult transgenic BP1 founder mouse among 121 mice was obtained. This mouse presumably survived due to transgene mosaicism because the transgene could not be transmitted. This mouse expressed BP1 and displayed splenomegaly, extramedullary erythropoiesis and severe amyloidosis A in the kidney, a phenotype compatible with thalassemia. Consistently, the presence of BP1 transgene in fetuses was associated with paleness and lethality. In ES cells, BP1 expression in primary differentiation appeared to antagonize adult beta-globin expression. In secondary differentiation, BP1 expression reduced significantly beta-globin gene expression in both primitive and definitive erythroid cells, whereas it impaired only the definitive erythroid cell differentiation. These studies showed that BP1 can negatively modulate adult beta-globin gene expression and definitive erythroid cell differentiation, and suggest that BP1 could play a role in thalassemia.