RESUMEN
White matter injuries (WMIs) are the leading cause of neurologic impairment in infants born premature. There are no treatment options available. The most common forms of WMIs in infants occur prior to the onset of normal myelination, making its pathophysiology distinctive, thus requiring a tailored approach to treatment. Neonates present a unique opportunity to repair WMIs due to a transient abundance of neural stem/progenitor cells (NSPCs) present in the germinal matrix with oligodendrogenic potential. We identified an endogenous oxysterol, 20-αHydroxycholesterol (20HC), in human maternal breast milk that induces oligodendrogenesis through a sonic hedgehog (shh), Gli-dependent mechanism. Following WMI in neonatal mice, injection of 20HC induced subventricular zone-derived oligodendrogenesis and improved myelination in the periventricular white matter, resulting in improved motor outcomes. Targeting the oligodendrogenic potential of postnatal NSPCs in neonates with WMIs may be further developed into a novel approach to mitigate this devastating complication of preterm birth.
Asunto(s)
Lesiones Encefálicas , Nacimiento Prematuro , Sustancia Blanca , Femenino , Humanos , Animales , Ratones , Recién Nacido , Sustancia Blanca/metabolismo , Leche Humana/metabolismo , Proteínas Hedgehog/metabolismo , Ventrículos Cerebrales/metabolismo , Oligodendroglía/fisiologíaRESUMEN
Background & Aims: Multicopper ferroxidases (MCFs) facilitate intestinal iron absorption and systemic iron recycling, likely by a mechanism involving the oxidization of Fe2+ from the iron exporter ferroportin 1 for delivery to the circulating Fe3+ carrier transferrin. Hephaestin (HEPH), the only MCF known to be expressed in enterocytes, aids in the basolateral transfer of dietary iron to the blood. Mice lacking HEPH in the whole body (Heph-/- ) or intestine alone (Hephint/int ) exhibit defects in dietary iron absorption but still survive and grow. Circulating ceruloplasmin (CP) is the only other known MCF likely to interact with enterocytes. Our aim was to assess the effects of combined deletion of HEPH and CP on intestinal iron absorption and homeostasis in mice. Methods: Mice lacking both HEPH and CP (Heph-/-Cp-/- ) and mice with whole-body knockout of CP and intestine-specific deletion of HEPH (Hephint/intCp-/- ) were generated and phenotyped. Results: Heph-/-Cp-/- mice were severely anemic and had low serum iron, but they exhibited marked iron loading in duodenal enterocytes, the liver, heart, pancreas, and other tissues. Hephint/intCp-/- mice were moderately anemic (similar to Cp-/- mice) but were iron loaded only in the duodenum and liver, as in Hephint/int and Cp-/- mice, respectively. Both double knockout models absorbed iron in radiolabeled intestinal iron absorption studies, but the iron was inappropriately distributed, with an abnormally high percentage retained in the liver. Conclusions: These studies indicate that HEPH and CP, and likely MCFs in general, are not essential for intestinal iron absorption but are required for proper systemic iron distribution. They also point to important extra-intestinal roles for HEPH in maintaining whole-body iron homeostasis.
Asunto(s)
Ceruloplasmina/deficiencia , Hierro/metabolismo , Proteínas de la Membrana/deficiencia , Absorción Fisiológica , Anemia/patología , Animales , Animales Lactantes , Tamaño Corporal , Peso Corporal , Proteínas de Transporte de Catión/metabolismo , Ceruloplasmina/metabolismo , Modelos Animales de Enfermedad , Duodeno/metabolismo , Enterocitos/metabolismo , Femenino , Ligadura , Masculino , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , FenotipoRESUMEN
Birth defects of the heart and face are common, and most have no known genetic cause, suggesting a role for environmental factors. Maternal fever during the first trimester is an environmental risk factor linked to these defects. Neural crest cells are precursor populations essential to the development of both at-risk tissues. We report that two heat-activated transient receptor potential (TRP) ion channels, TRPV1 and TRPV4, were present in neural crest cells during critical windows of heart and face development. TRPV1 antagonists protected against the development of hyperthermia-induced defects in chick embryos. Treatment with chemical agonists of TRPV1 or TRPV4 replicated hyperthermia-induced birth defects in chick and zebrafish embryos. To test whether transient TRPV channel permeability in neural crest cells was sufficient to induce these defects, we engineered iron-binding modifications to TRPV1 and TRPV4 that enabled remote and noninvasive activation of these channels in specific cellular locations and at specific developmental times in chick embryos with radio-frequency electromagnetic fields. Transient stimulation of radio frequency-controlled TRP channels in neural crest cells replicated fever-associated defects in developing chick embryos. Our data provide a previously undescribed mechanism for congenital defects, whereby hyperthermia activates ion channels that negatively affect fetal development.
Asunto(s)
Anomalías Congénitas/etiología , Fiebre/complicaciones , Insuficiencia Cardíaca/etiología , Cresta Neural/patología , Canales Catiónicos TRPV/metabolismo , Animales , Embrión de Pollo , Pollos , Anomalías Congénitas/metabolismo , Anomalías Congénitas/patología , Femenino , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Intercambio Materno-Fetal , Ratones , Ratones Endogámicos C57BL , Cresta Neural/metabolismo , Embarazo , Pez CebraRESUMEN
Despite the accelerated discovery of genes associated with syndromic traits, the majority of families affected by such conditions remain undiagnosed. Here, we employed whole-exome sequencing in two unrelated consanguineous kindreds with central nervous system (CNS), cardiac, renal, and digit abnormalities. We identified homozygous truncating mutations in TMEM260, a locus predicted to encode numerous splice isoforms. Systematic expression analyses across tissues and developmental stages validated two such isoforms, which differ in the utilization of an internal exon. The mutations in both families map uniquely to the long isoform, raising the possibility of an isoform-specific disorder. Consistent with this notion, RT-PCR of lymphocyte cell lines from one of the kindreds showed reduced levels of only the long isoform, which could be ameliorated by emetine, suggesting that the mutation induces nonsense-mediated decay. Subsequent in vivo testing supported this hypothesis. First, either transient suppression or CRISPR/Cas9 genome editing of zebrafish tmem260 recapitulated key neurological phenotypes. Second, co-injection of morphants with the long human TMEM260 mRNA rescued CNS pathology, whereas the short isoform was significantly less efficient. Finally, immunocytochemical and biochemical studies showed preferential enrichment of the long TMEM260 isoform to the plasma membrane. Together, our data suggest that there is overall reduced, but not ablated, functionality of TMEM260 and that attenuation of the membrane-associated functions of this protein is a principal driver of pathology. These observations contribute to an appreciation of the roles of splice isoforms in genetic disorders and suggest that dissection of the functions of these transcripts will most likely inform pathomechanism.
Asunto(s)
Anomalías Múltiples/genética , Síndrome Cardiorrenal/genética , Proteínas de la Membrana/genética , Trastornos del Neurodesarrollo/genética , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Linaje , Mutación Puntual , Isoformas de Proteínas/genéticaRESUMEN
Disrupted brain iron homeostasis is a common feature of neurodegenerative disease. To begin to understand how neuronal iron handling might be involved, we focused on dopaminergic neurons and asked how inactivation of transport proteins affected iron homeostasis in vivo in mice. Loss of the cellular iron exporter, ferroportin, had no apparent consequences. However, loss of transferrin receptor 1, involved in iron uptake, caused neuronal iron deficiency, age-progressive degeneration of a subset of dopaminergic neurons, and motor deficits. There was gradual depletion of dopaminergic projections in the striatum followed by death of dopaminergic neurons in the substantia nigra. Damaged mitochondria accumulated, and gene expression signatures indicated attempted axonal regeneration, a metabolic switch to glycolysis, oxidative stress, and the unfolded protein response. We demonstrate that loss of transferrin receptor 1, but not loss of ferroportin, can cause neurodegeneration in a subset of dopaminergic neurons in mice.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Hierro/metabolismo , Degeneración Nerviosa/etiología , Degeneración Nerviosa/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Neuronas Dopaminérgicas/patología , Femenino , Homeostasis , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Degeneración Nerviosa/patología , Receptores de Transferrina/deficiencia , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismoRESUMEN
Helicobacter pylori infection triggers chronic inflammation of the gastric mucosa that may progress to gastric cancer. The hypoxia-inducible factors (HIFs) are the central mediators of cellular adaptation to low oxygen levels (hypoxia), but they have emerged recently as major transcriptional regulators of immunity and inflammation. No studies have investigated whether H. pylori affects HIF signaling in immune cells and a potential role for HIF in H. pylori-mediated gastritis. HIF-1 and HIF-2 expression was examined in human H. pylori-positive gastritis biopsies. Subsequent experiments were performed in naive and polarized bone marrow-derived macrophages from wild-type (WT) and myeloid HIF-1α-null mice (HIF-1(Δmyel)). WT and HIF-1(Δmyel) mice were inoculated with H. pylori by oral gavage and sacrificed 6 mo postinfection. HIF-1 was specifically expressed in macrophages of human H. pylori-positive gastritis biopsies. Macrophage HIF-1 strongly contributed to the induction of proinflammatory genes (IL-6, IL-1ß) and inducible NO synthase in response to H. pylori. HIF-2 expression and markers of M2 macrophage differentiation were decreased in response to H. pylori. HIF-1(Δmyel) mice inoculated with H. pylori for 6 mo presented with a similar bacterial colonization than WT mice but, surprisingly, a global increase of inflammation, leading to a worsening of the gastritis, measured by an increased epithelial cell proliferation. In conclusion, myeloid HIF-1 is protective in H. pylori-mediated gastritis, pointing to the complex counterbalancing roles of innate immune and inflammatory phenotypes in driving this pathology.
Asunto(s)
Gastritis/etiología , Gastritis/metabolismo , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/metabolismo , Helicobacter pylori , Factor 1 Inducible por Hipoxia/metabolismo , Células Mieloides/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biopsia , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Mucosa Gástrica/inmunología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Gastritis/patología , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/inmunología , Helicobacter pylori/inmunología , Humanos , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Transgénicos , Células Mieloides/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/microbiologíaRESUMEN
Although earlier, seminal studies demonstrated that the gut per se has the intrinsic ability to regulate the rates of iron absorption, the spotlight in the past decade has been placed on the systemic regulation of iron homeostasis by the hepatic hormone hepcidin and the molecular mechanisms that regulate its expression. Recently, however, attention has returned to the gut based on the finding that hypoxia inducible factor-2 (HIF-2α) regulates the expression of key genes that contribute to iron absorption. Here we review the current understanding of the molecular mechanisms that regulate iron homeostasis in the gut by focusing on the role of HIF-2 under physiological steady-state conditions and in the pathogenesis of iron-related diseases. We also discuss implications for adapting HIF-2-based therapeutic strategies in iron-related pathological conditions.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/fisiopatología , Regulación de la Expresión Génica , Hierro/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Enterocitos/citología , Hemocromatosis/metabolismo , Hemocromatosis/fisiopatología , Hepatocitos/metabolismo , Hepcidinas , Homeostasis , Humanos , Hipoxia , Inflamación/metabolismo , Inflamación/fisiopatología , Proteína 1 Reguladora de Hierro/metabolismo , Proteína 2 Reguladora de Hierro/metabolismo , Masculino , Ratones , Policitemia/metabolismo , Policitemia/fisiopatologíaRESUMEN
Iron and copper are essential trace metals, actively absorbed from the proximal gut in a regulated fashion. Depletion of either metal can lead to anemia. In the gut, copper deficiency can affect iron absorption through modulating the activity of hephaestin - a multi-copper oxidase required for optimal iron export from enterocytes. How systemic copper status regulates iron absorption is unknown. Mice were subjected to a nutritional copper deficiency-induced anemia regime from birth and injected with copper sulphate intraperitoneally to correct the anemia. Copper deficiency resulted in anemia, increased duodenal hypoxia and Hypoxia inducible factor 2α (HIF-2α) levels, a regulator of iron absorption. HIF-2α upregulation in copper deficiency appeared to be independent of duodenal iron or copper levels and correlated with the expression of iron transporters (Ferroportin - Fpn, Divalent Metal transporter - Dmt1) and ferric reductase - Dcytb. Alleviation of copper-dependent anemia with intraperitoneal copper injection resulted in down regulation of HIF-2α-regulated iron absorption genes in the gut. Our work identifies HIF-2α as an important regulator of iron transport machinery in copper deficiency.
Asunto(s)
Anemia/etiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Cobre/deficiencia , Duodeno/patología , Hierro/metabolismo , Regulación hacia Arriba , Absorción/efectos de los fármacos , Absorción/genética , Anemia/tratamiento farmacológico , Animales , Hipoxia de la Célula/efectos de los fármacos , Cobre/administración & dosificación , Cobre/farmacología , Cobre/uso terapéutico , Regulación hacia Abajo/efectos de los fármacos , Duodeno/efectos de los fármacos , Femenino , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos C57BL , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Hypoxia, via stabilization of HIF2α, regulates the expression of the intestinal iron transporters DMT1 and ferroportin. Here we investigated whether the intestinal copper importer Ctr1 was also regulated by hypoxia. Copper uptake and Ctr1 mRNA expression were significantly increased in Caco-2 cells exposed to hypoxia. To determine whether HIF2α was involved in regulation of Ctr1 expression, we employed three models of HIF2α knockdown (chemical suppression of HIF2α translation in Caco-2 cells; HIF2α-siRNA-treated HuTu80 cells; HIF2α-intestinal knockout mice); Ctr1 mRNA expression was decreased in all three models under normoxic conditions. HIF2α translational inhibitor did not alter Ctr1 expression under hypoxic conditions. We conclude that basal expression of Ctr1 is regulated by HIF2α; however, the induction by hypoxia is a HIF2α-independent event.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Mucosa Intestinal/metabolismo , Animales , Células CACO-2 , Proteínas de Transporte de Catión/metabolismo , Línea Celular Tumoral , Cobre/farmacología , Transportador de Cobre 1 , Duodeno/metabolismo , Homeostasis , Humanos , Hipoxia , Hierro/farmacología , Ratones , Ratones NoqueadosRESUMEN
Mutations of the TMPRSS6 gene, which encodes Matriptase-2, are responsible for iron-refractory iron-deficiency anemia. Matriptase-2 is a transmembrane protease that downregulates hepcidin expression. We report one frameshift (p.Ala605ProfsX8) and four novel missense mutations (p.Glu114Lys, p.Leu235Pro, p.Tyr418Cys, p.Pro765Ala) found in IRIDA patients. These mutations lead to changes in both the catalytic and noncatalytic domains of Matriptase-2. Analyses of the mutant proteins revealed a reduction of autoactivating cleavage and the loss of N-Boc-Gln-Ala-Arg-p-nitroanilide hydrolysis. This resulted either from a direct modification of the active site or from the lack of the autocatalytic cleavage that transforms the zymogen into an active protease. In a previously described transfection assay measuring the ability of Matriptase-2 to repress the hepcidin gene (HAMP) promoter, all mutants retained some, if not all, of their transcriptional repression activity. This suggests that caution is called for in interpreting the repression assay in assessing the functional relevance of Matriptase-2 substitutions. We propose that Matriptase-2 activity should be measured directly in the cell medium of transfected cells using the chromogenic substrate. This simple test can be used to determine whether a sequence variation leading to an amino acid substitution is functionally relevant or not.
Asunto(s)
Anemia Ferropénica/enzimología , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Serina Proteasas/metabolismo , Transfección/métodos , Adolescente , Adulto , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Anemia Ferropénica/genética , Péptidos Catiónicos Antimicrobianos/sangre , Péptidos Catiónicos Antimicrobianos/genética , Dominio Catalítico , Niño , Preescolar , Compuestos Cromogénicos/metabolismo , Medios de Cultivo/metabolismo , Activación Enzimática , Pruebas de Enzimas , Precursores Enzimáticos/metabolismo , Mutación del Sistema de Lectura , Silenciador del Gen , Pruebas Genéticas , Células HeLa , Hepcidinas , Humanos , Lactante , Masculino , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación Missense , Linaje , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Serina Endopeptidasas/genética , Transcripción GenéticaRESUMEN
Hereditary hemochromatosis (HH) is a highly prevalent genetic disorder characterized by excessive parenchymal iron accumulation leading to liver cirrhosis, diabetes, and in some cases hepatocellular carcinoma. HH is caused by mutations in the genes encoding upstream regulators of hepcidin or more rarely in the hepcidin gene itself. A deficit in hepcidin results in intestinal iron hyperabsorption; however, the local effectors mediating the up-regulation of iron absorption genes are unknown. We hypothesized that HIF-2 could mediate high iron absorption rates in HH. We generated Hepc(-/-) mice (a murine model of hemochromatosis) lacking HIF-2 in the intestine and showed that duodenal HIF-2 was essential for the up-regulation of genes involved in intestinal iron import and the consequent iron accumulation in the liver and pancreas. This study highlights a role of HIF-2 in the dysregulation of iron absorption and chronic iron accumulation, as observed in patients with hemochromatosis.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Enterocitos/metabolismo , Mucosa Intestinal/metabolismo , Sobrecarga de Hierro/prevención & control , Animales , Western Blotting , Duodeno/metabolismo , Duodeno/patología , Enterocitos/patología , Femenino , Hemocromatosis/etiología , Hepcidinas , Técnicas para Inmunoenzimas , Integrasas/metabolismo , Intestinos/patología , Sobrecarga de Hierro/etiología , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The BMP/SMAD4 pathway has major effects on liver hepcidin levels. Bone morphogenetic protein-binding endothelial cell precursor-derived regulator (Bmper), a known regulator of BMP signaling, was found to be overexpressed at the mRNA and protein levels in liver of genetically hypotransferrinemic mice (Trf(hpx/hpx)). Soluble BMPER peptide inhibited BMP2- and BMP6-dependent hepcidin promoter activity in both HepG2 and HuH7 cells. These effects correlated with reduced cellular levels of pSMAD1/5/8. Addition of BMPER peptide to primary human hepatocytes abolished the BMP2-dependent increase in hepcidin mRNA, whereas injection of Bmper peptide into mice resulted in reduced liver hepcidin and increased serum iron levels. Thus Bmper may play an important role in suppressing hepcidin production in hypotransferrinemic mice.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/sangre , Proteínas Portadoras/metabolismo , Hierro/sangre , Hígado/metabolismo , Transferrina/metabolismo , Regulación hacia Arriba , Animales , Péptidos Catiónicos Antimicrobianos/genética , Proteína Morfogenética Ósea 2/antagonistas & inhibidores , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Proteínas Portadoras/genética , Células Hep G2 , Hepcidinas , Humanos , Ratones , Ratones Transgénicos , Péptidos/farmacología , Proteínas Smad/genética , Proteínas Smad/metabolismo , Transferrina/genéticaRESUMEN
BACKGROUND: Iron metabolism, regulated by the iron hormone hepcidin, and oxygen homeostasis, dependent on hypoxia-inducible factors, are strongly interconnected. We previously reported that in mice in which both liver hypoxia-inducible factors-1 and -2 are stabilized (the hepatocyte von Hippel-Lindau knockout mouse model), hepcidin expression was strongly repressed and we hypothesized that hypoxia-inducible factor-2 could be the major regulatory component contributing to the hepcidin down-regulation. DESIGN AND METHODS: We generated and analyzed hepatocyte-specific knockout mice harboring either hypoxia-inducible factor-2α deficiency (Hif2a knockout) or constitutive hypoxia-inducible factor-2α stabilization (Vhlh/Hif1a knockout) and ex vivo systems (primary hepatocyte cultures). Hif2a knockout mice were fed an iron-deficient diet for 2 months and Vhlh/Hif1a knockout mice were treated with neutralizing erythropoietin antibody. RESULTS: We demonstrated that hypoxia-inducible factor-2 is dispensable in hepcidin gene regulation in the context of an adaptive response to iron-deficiency anemia. However, its overexpression in the double Vhlh/Hif1a hepatocyte-specific knockout mice indirectly down-regulates hepcidin expression through increased erythropoiesis and erythropoietin production. Experiments in primary hepatocytes confirmed the non-autonomous role of hypoxia-inducible factor-2 in hepcidin regulation. CONCLUSIONS: While our results indicate that hypoxia-inducible factor-2 is not directly involved in hepcidin repression, they highlight the contribution of hepatic hypoxia-inducible factor-2 to the repression of hepcidin through erythropoietin-mediated increased erythropoiesis, a result of potential clinical interest.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Eritropoyetina/genética , Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Animales , Anticuerpos Neutralizantes/farmacología , Péptidos Catiónicos Antimicrobianos/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Eritropoyesis/efectos de los fármacos , Eritropoyesis/fisiología , Eritropoyetina/antagonistas & inhibidores , Femenino , Alimentos Formulados , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepcidinas , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Deficiencias de Hierro , Masculino , Ratones , Ratones Noqueados , Cultivo Primario de Células , Transducción de Señal/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Based on a microarray study, which demonstrated the upregulation of Sp6 transcriptional factor in iron deficient rat duodenum, we reasoned that SP6 could regulate iron absorption by controlling the expression of iron absorption genes (Collins,2006). For that, we generated Sp6 specific intestinal knockout mice. Our data suggest a lack of transcriptional upregulation of Sp6 in mice in conditions where iron absorption is promoted (phlebotomy, iron deficiency, hpx mouse), and an absence of iron-related phenotype in Sp6 intestinal knockout model. We propose that other Sp6 orthologues may be involved in the genetic response to increase iron absorption, possibly in co-operation with hypoxia inducible factor 2 alpha (HIF-2α)-a newly discovered regulator of iron absorption.
Asunto(s)
Mucosa Intestinal/metabolismo , Hierro/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Fenotipo , Anemia Ferropénica/genética , Anemia Ferropénica/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Transporte de Catión/genética , Regulación de la Expresión Génica , Deficiencias de Hierro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras Genéticas/genética , Activación Transcripcional/genéticaRESUMEN
Hepcidin negatively regulates systemic iron homeostasis in response to inflammation and elevated serum iron. Conversely, hepcidin expression is diminished in response to hypoxia, oxidative stress, and increased erythropoietic demand, though the molecular intermediates involved are incompletely understood. To address this, we have investigated hypoxic hepcidin regulation in HuH7 hepatoma cells either cultured alone or cocultured with activated THP-1 macrophages. HuH7 hepcidin mRNA expression was determined using quantitative polymerase chain reaction (Q-PCR). Hepcidin promoter activity was measured using luciferase reporter constructs containing a 0.9 kb fragment of the wild-type human hepcidin promoter, and constructs containing mutations in bone morphogenetic protein (BMP)/SMAD4, signal transducer and activator of transcription 3 (STAT3), CCAAT/enhancer-binding protein (C/EBP), and E-box-responsive elements. Hepatic expression of bone morphogenetic proteins BMP2 and BMP6 and the BMP inhibitor noggin was determined using Q-PCR, and the protein expression of hemojuvelin (HJV), pSMAD 1/5/8, and SMAD4 was determined by western blotting. Following exposure to hypoxia or H(2)O(2), hepcidin mRNA expression and promoter activity increased in HuH7 cells monocultures but were decreased in HuH7 cells cocultured with THP-1 macrophages. This repression was attenuated by mutation of the BMP/SMAD4-response element, suggesting that modulation of SMAD signaling mediated the response to hypoxia. No changes in hepatocyte BMP2, BMP6 or noggin mRNA, or protein expression of HJV or pSMAD 1/5/8 were detected. However, treatment with hypoxia caused a marked decrease in nuclear and cytosolic SMAD4 protein and SMAD4 mRNA expression in cocultured HuH7 cells. Together these data indicate that hypoxia represses hepcidin expression through inhibition of BMP/SMAD signaling.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Hipoxia/metabolismo , Transducción de Señal/fisiología , Proteína Smad4/metabolismo , Péptidos Catiónicos Antimicrobianos/genética , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Medios de Cultivo Condicionados/metabolismo , Hepcidinas , Humanos , Neoplasias Hepáticas/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Mutación , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Erythropoietic activity is known to affect iron homeostasis through regulation of the liver iron regulatory hormone hepcidin. To identify new factors secreted by the erythroblasts that could influence hepcidin synthesis, we set up a coculture model. HuH7 hepatoma cells cocultured with primary human erythroblasts or erythroleukemic UT7 cells presented a 20- to 35-fold increase of hepcidin gene expression. This induction was fully blunted in the presence of a neutralizing oncostatin M antibody, demonstrating that this cytokine, belonging to the IL-6 family of cytokines, was responsible for increased levels of hepcidin expression. We further demonstrated that recombinant oncostatin M induced a dramatic transcriptional increase of hepcidin in HuH7 cells through specific activation of the STAT pathway. Hepcidin induction by oncostatin M was also observed in hepatocytes in primary culture and is believed to be cell specific since no induction was found in isolated bone marrow cells, macrophagic, stromal, and lymphoma-derived cell lines, nor in erythroblasts. Finally, we show that oncostatin M administration in vivo increases hepcidin expression and leads to significantly decreased serum iron levels. This work identifies a new potent inducer of hepcidin expression in the liver and supports a role for modulators of oncostatin M signaling pathway in treating iron disorders.
Asunto(s)
Antibacterianos/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , Antineoplásicos/farmacología , Carcinoma Hepatocelular/metabolismo , Eritroblastos/metabolismo , Hierro/metabolismo , Neoplasias Hepáticas/metabolismo , Oncostatina M/farmacología , Animales , Antineoplásicos/antagonistas & inhibidores , Antineoplásicos/inmunología , Western Blotting , Carcinoma Hepatocelular/patología , Células Cultivadas , Técnicas de Cocultivo , Hepcidinas , Humanos , Interleucina-6/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Oncostatina M/antagonistas & inhibidores , Oncostatina M/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción STAT/metabolismoRESUMEN
BACKGROUND: Hepcidin is an iron regulatory peptide produced by the liver in response to inflammation and elevated systemic iron. Recent studies suggest that circulating monocytes and resident liver macrophages--Küpffer cells--may influence both basal and inflammatory expression of hepcidin. DESIGN AND METHODS: We used an in vitro co-culture model to investigate hepatocyte hepcidin regulation in the presence of activated THP1 macrophages. HuH7 hepatoma cells were co-cultured with differentiated THP1 macrophages for 24 h prior to the measurement of HuH7 hepcidin (HAMP) mRNA expression using quantitative polymerase chain reaction, and HAMP promoter activity using a luciferase reporter assay. Luciferase assays were performed using the wild type HAMP promoter, and constructs containing mutations in BMP/SMAD4, STAT3, C/EBP and E-BOX response elements. Neutralizing antibodies against interleukin-6, interleukin-1beta , and the bone morphogenetic protein inhibitor noggin were used to identify the macrophage-derived cytokines involved in the regulation of HAMP expression. RESULTS: Co-culturing HuH7 cells with differentiated THP1 cells induced HAMP promoter activity and endogenous HAMP mRNA expression maximally after 24 h. This induction was fully neutralized in the presence of an interleukin-1beta antibody, and fully attenuated by mutations of the proximal C/EBP or BMP/SMAD4 response elements. CONCLUSIONS: Our data suggest that the interleukin-1beta and bone morphogenetic protein signaling pathways are central to the regulation of HAMP expression by macrophages in this co-culture model.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/genética , Regulación Neoplásica de la Expresión Génica/genética , Macrófagos/metabolismo , Anticuerpos/farmacología , Sitios de Unión/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Línea Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hepcidinas , Humanos , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/inmunología , Interleucina-6/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Activación de Macrófagos , Macrófagos/citología , Monocitos/citología , Monocitos/metabolismo , Mutación , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Smad4/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
HIF transcription factors (HIF-1 and HIF-2) are central mediators of cellular adaptation to hypoxia. Because the resting partial pressure of oxygen is low in the intestinal lumen, epithelial cells are believed to be mildly hypoxic. Having recently established a link between HIF and the iron-regulatory hormone hepcidin, we hypothesized that HIFs, stabilized in the hypoxic intestinal epithelium, may also play critical roles in regulating intestinal iron absorption. To explore this idea, we first established that the mouse duodenum, the site of iron absorption in the intestine, is hypoxic and generated conditional knockout mice that lacked either Hif1a or Hif2a specifically in the intestinal epithelium. Using these mice, we found that HIF-1alpha was not necessary for iron absorption, whereas HIF-2alpha played a crucial role in maintaining iron balance in the organism by directly regulating the transcription of the gene encoding divalent metal transporter 1 (DMT1), the principal intestinal iron transporter. Specific deletion of Hif2a led to a decrease in serum and liver iron levels and a marked decrease in liver hepcidin expression, indicating the involvement of an induced systemic response to counteract the iron deficiency. This finding may provide a basis for the development of new strategies, specifically in targeting HIF-2alpha, to improve iron homeostasis in patients with iron disorders.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Regulación de la Expresión Génica/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Absorción Intestinal/fisiología , Hierro/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/genética , Células CACO-2 , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Hipoxia de la Célula/fisiología , Citocromos b/genética , Duodeno/metabolismo , Eritrocitos/química , Eritrocitos/citología , Expresión Génica/genética , Hemoglobinas/análisis , Hepcidinas , Humanos , Hierro/sangre , Deficiencias de Hierro , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regiones Promotoras Genéticas/fisiología , Unión Proteica/genética , Elementos de Respuesta/fisiología , Activación Transcripcional/fisiologíaRESUMEN
Obesity is a major global health problem and is associated with low-grade inflammation and, in a number of cases, poor iron status. We speculated that the adipokine leptin might play a role in regulating iron metabolism in the overweight population because it shares a number of common biological features with IL-6, a major factor in the development of the anemia of chronic disease via its stimulatory actions on the production and release of the iron regulatory hormone hepcidin. To test this hypothesis, we exposed HuH7 human hepatoma cells to leptin and measured hepcidin mRNA expression by quantitative PCR. HuH7 cells were also transfected with a hepcidin promoter-luciferase reporter gene construct to investigate transcriptional regulation of hepcidin. In leptin-treated cells, hepcidin mRNA expression was enhanced significantly. Preincubation with a Janus kinase (JAK) 2 inhibitor significantly diminished this response. Hepcidin promoter activity was also increased in the presence of leptin. This effect was decreased either by mutation of the signal transducer and activator of transcription (STAT) 3 binding motif in the hepcidin promoter or by coexpressing a dominant-negative STAT3 mutant. These data suggest that leptin upregulates hepatic hepcidin expression through the JAK2/STAT3 signaling pathway. As a consequence, the increased production of leptin in overweight individuals might be a major contributor to the aberrant iron status observed in these population groups.