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 potassium chloride cotransporters (KCC) family of proteins are widely expressed and are involved in the transepithelial movement of potassium and chloride ions and the regulation of cell volume. KCC activity is high in reticulocytes, and contributes to the dehydration of sickle red blood cells. Because plasma levels of both vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are elevated in sickle cell individuals, and VEGF has been shown to increase KCC expression in other cells, we hypothesized that VEGF and PlGF influence KCC expression in erythroid cells. Both VEGF and PlGF treatment of human erythroid K562 cells increased both mRNA and protein levels of KCC1, KCC3b, and KCC4. VEGF- and PlGF-mediated cellular signaling involved VEGF-R1 and downstream effectors, specifically, PI-3 kinase, p38 MAP kinase, mTOR, NADPH-oxidase, JNK kinase, and HIF-1α. VEGF and PlGF-mediated transcription of KCC3b and KCC4 involved hypoxia response element (HRE) motifs in their promoters, as demonstrated by promoter analysis, EMSA and ChiP. These results were corroborated in vivo by adenoviral-mediated overexpression of PlGF in normal mice, which led to increased expression of mKCC3 and mKCC4 in erythroid precursors. Our studies show that VEGF and PlGF regulate transcription of KCC3b and KCC4 in erythroid cells via activation of HIF-1α, independent of hypoxia. These studies provide novel therapeutic targets for regulation of cell volume in RBC precursors, and thus, amelioration of dehydration in RBCs in sickle cell disease.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Proteínas Gestacionales/fisiología , Simportadores/biosíntesis , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Hipoxia de la Célula , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Células K562 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Factor de Crecimiento Placentario , Proteínas Gestacionales/genética , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , ARN Interferente Pequeño/farmacología , Simportadores/genética , Transcripción Genética , Transducción Genética , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Receptor 1 de Factores de Crecimiento Endotelial Vascular/fisiología , Cotransportadores de K ClRESUMEN
Hypoxia occurs in a number of pathological states, such as pulmonary, hematological, and cardiovascular disorders. In this study, we examined the molecular mechanism by which hypoxia contributes to increased leukotriene formation. Our studies showed hypoxia augmented the expression of 5-lipoxygenase activating protein (FLAP), a key enzyme in leukotriene formation, in both human pulmonary microvascular endothelial cells and a transformed human brain endothelial cell line. Hypoxia-induced FLAP mRNA expression involved activation of NADPH-oxidase, PI-3 kinase, mitogen-activated protein kinase, NF-kappaB, and hypoxia-inducible factor (HIF)-1alpha. Hypoxia-induced FLAP promoter activity was attenuated on mutation of hypoxia-response elements (HREs) and NF-kappaB binding motif in the FLAP promoter. Hypoxia also augmented binding of HIF-1alpha to HREs in FLAP promoter as demonstrated by EMSA with nuclear extracts. Furthermore, chromain immunoprecipitation analysis showed HIF-1alpha bound to HREs in native chromatin obtained from hypoxia-treated cells. Next, we examined the role of HIF-1alpha regulated microRNAs on FLAP expression. Our studies showed decreased expression of miR-135a and miR-199a-5p in response to hypoxia. However, overexpression of anti-miR-135a and anti-miR-199a-5p oligonucleotides led to a several fold increased FLAP mRNA and protein expression. These studies demonstrate for the first time that hypoxia-mediated FLAP expression is regulated by HREs and NF-kappaB site in its promoter, and negatively regulated by miR-135a and miR-199a-5p, which target the 3'-UTR of FLAP mRNA. An understanding of these regulatory pathways provides new avenues to ameliorate leukotriene formation and hence reactive airway disease, and inflammation in individuals who have sickle cell disease.
Asunto(s)
Proteínas Portadoras/biosíntesis , Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Proteínas de la Membrana/biosíntesis , MicroARNs/metabolismo , FN-kappa B/metabolismo , Proteínas Activadoras de la 5-Lipooxigenasa , Western Blotting , Ensayo de Cambio de Movilidad Electroforética , Células Endoteliales/enzimología , Expresión Génica , Humanos , Mutagénesis Sitio-Dirigida , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Individuals with sickle cell disease (SCD) have increased inflammation, a high incidence of airway hyperreactivity (AH), and increased circulating leukotrienes (LT). We show that expression of 5-lipoxygenase and 5-lipoxygenase activating protein (FLAP), key catalytic molecules in the LT pathway, were significantly increased in peripheral blood mononuclear cells (MNCs) in patients with SCD, compared with healthy controls. Placenta growth factor (PlGF), elaborated from erythroid cells, activated MNC and THP-1 monocytic cells to induce LT production. PlGF-mediated increased FLAP mRNA expression occurred via activation of phosphoinositide-3 (PI-3) kinase, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and hypoxia inducible factor-1alpha (HIF-1alpha). HIF-1alpha small interfering RNA (siRNA) reduced PlGF-induced FLAP expression. FLAP promoter-driven luciferase constructs demonstrated that PlGF-mediated luciferase induction was abrogated upon mutation of HIF-1alpha response element (HRE), but not the nuclear factor-kappaB (NF-kappaB) site in the FLAP promoter; a finding confirmed by chromatin immunoprecipitation (ChIP) analysis. PlGF also increased HIF-1alpha binding to the HRE in the FLAP promoter. Therefore, it is likely that the intrinsically elevated levels of PlGF in SCD subjects contribute to increased LT, which in turn, mediate both inflammation and AH. Herein, we identify a mechanism of increased LT in SCD and show HIF-1alpha as a hypoxia-independent target of PlGF. These studies provide new avenues to ameliorate these complications.
Asunto(s)
Anemia de Células Falciformes/metabolismo , Proteínas Portadoras/biosíntesis , Regulación de la Expresión Génica , Leucotrienos/biosíntesis , Proteínas de la Membrana/biosíntesis , Proteínas Gestacionales/metabolismo , Hipersensibilidad Respiratoria/metabolismo , Proteínas Activadoras de la 5-Lipooxigenasa , Anemia de Células Falciformes/complicaciones , Araquidonato 5-Lipooxigenasa/metabolismo , Línea Celular , Niño , Preescolar , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , NADPH Oxidasas/metabolismo , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Crecimiento Placentario , Hipersensibilidad Respiratoria/etiología , Elementos de RespuestaRESUMEN
Pulmonary hypertension (PHT) develops in sickle cell disease (SCD) and is associated with high mortality. We previously showed that erythroid cells produce placenta growth factor (PlGF), which activates monocytes to induce proinflammatory cytochemokines, contributing to the baseline inflammation and severity in SCD. In this study, we observed that PlGF increased expression of endothelin-1 (ET-1) and endothelin-B receptor (ET-BR) from human pulmonary microvascular endothelial cells (HPMVECs) and monocytes, respectively. PlGF-mediated ET-1 and ET-BR expression occurred via activation of PI-3 kinase, reactive oxygen species and hypoxia inducible factor-1 alpha (HIF-1 alpha). PlGF increased binding of HIF-1 alpha to the ET-1 and ET-BR promoters; this effect was abrogated with mutation of hypoxia response elements in the promoter regions and HIF-1 alpha siRNA and confirmed by chromatin immunoprecipitation analysis. Furthermore, PlGF-mediated ET-1 release from HPMVECs and ET-BR expression in monocytes creates a PlGF-ET-1-ET-BR loop, leading to increased expression of MCP-1 and IL-8. Our studies show that PlGF-induced expression of the potent vasoconstrictor ET-1 and its cognate ET-BR receptor occur via activation of HIF-1 alpha, independent of hypoxia. PlGF levels are intrinsically elevated from the increased red cell turnover in SCD and in other chronic anemia (eg, thalassemia) and may contribute to inflammation and PHT seen in these diseases.
Asunto(s)
Endotelina-1/genética , Endotelio Vascular/citología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas Gestacionales/fisiología , Receptor de Endotelina B/genética , Células Cultivadas , Quimiocina CCL2/biosíntesis , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Expresión Génica , Humanos , Interleucina-8/biosíntesis , Monocitos/metabolismo , Factor de Crecimiento Placentario , Circulación PulmonarRESUMEN
Endothelin-1 (ET-1) and plasminogen activator inhibitor-1 (PAI-1) play important roles in pulmonary hypertension (PH) in sickle cell disease (SCD). Our previous studies show higher levels of placenta growth factor (PlGF) in SCD correlate with increased plasma levels of ET-1, PAI-1, and other physiological markers of PH. PlGF-mediated ET-1 and PAI-1 expression occurs via activation of hypoxia-inducible factor-1α (HIF-1α). However, relatively little is understood regarding post-transcriptional regulation of PlGF-mediated expression of ET-1 and PAI-1. Herein, we show PlGF treatment of endothelial cells reduced levels of miR-301a and miR-454 from basal levels. In addition, both miRNAs targeted the 3'-UTRs of ET-1 and PAI-1 mRNAs. These results were corroborated in the mouse model of SCD [Berkeley sickle mice (BK-SS)] and in SCD subjects. Plasma levels of miR-454 in SCD subjects were significantly lower compared with unaffected controls, which correlated with higher plasma levels of both ET-1 and PAI-1. Moreover, lung tissues from BK-SS mice showed significantly reduced levels of pre-miR-301a and concomitantly higher levels of ET-1 and PAI-1. Furthermore, we show that miR-301a/miR-454 located in the spindle and kinetochore-associated protein-2 (SKA2) transcription unit was co-transcriptionally regulated by both HIF-1α and peroxisome proliferator-activated receptor-α (PPAR-α) as demonstrated by SKA2 promoter mutational analysis and ChIP. Finally we show that fenofibrate, a PPAR-α agonist, increased the expression of miR-301a/miR-454 and SKA2 in human microvascular endothelial cell line (HMEC) cells; the former were responsible for reduced expression of ET-1 and PAI-1. Our studies provide a potential therapeutic approach whereby fenofibrate-induced miR-301a/miR-454 expression can ameliorate PH and lung fibrosis by reduction in ET-1 and PAI-1 levels in SCD.
Asunto(s)
Anemia de Células Falciformes/genética , Proteínas Cromosómicas no Histona/biosíntesis , Endotelina-1/biosíntesis , Hipertensión Pulmonar/genética , MicroARNs/biosíntesis , PPAR alfa/genética , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/patología , Animales , Línea Celular , Proteínas Cromosómicas no Histona/genética , Endotelina-1/genética , Fenofibrato/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hipertensión Pulmonar/complicaciones , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , MicroARNs/genética , PPAR alfa/antagonistas & inhibidores , PPAR alfa/metabolismo , Factor de Crecimiento Placentario , Inhibidor 1 de Activador Plasminogénico/genética , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , Regiones Promotoras GenéticasRESUMEN
Pulmonary hypertension (PHT) is associated with high mortality in sickle cell anemia (SCA). Previously, we showed that elevated levels of placenta growth factor (PlGF) in SCA patients correlate with increased levels of the potent vasoconstrictor endothelin-1 (ET-1) and PHT. Moreover, PlGF induced the expression of ET-1 via hypoxia-inducible factor 1α. Here, we show a novel example of ET-1 posttranscriptional regulation by PlGF via action of microRNA 648 (miR-648), which is subject to transcriptional coregulation with its host gene, MICAL3 (microtubule-associated monooxygenase, calponin, and LIM domain containing 3gene). PlGF repressed expression of miR-648 in endothelial cells. Luciferase reporter assays using wild-type and mutant ET-1 3' untranslated region (UTR) constructs, and transfection of miR-648 mimics showed that miR-648 targets the 3' UTR of ET-1 mRNA. Since miR-648 is located in a 5'-proximal intron of MICAL3, we examined which of three potential promoters was responsible for its expression. The MICAL3 distal promoter (P1) was the predominant promoter used for transcription of pre-miR-648, and it was under positive control by PAX5 (paired box protein 5) transcription factor, as demonstrated by the loss and gain of function of PAX5 activity, and chromatin immunoprecipitation analysis. These studies provide a novel link wherein PlGF-mediated downregulation of PAX5 attenuates miR-648 expression leading to increased ET-1 levels that are known to induce PHT in SCA.