RESUMEN
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
Hepcidin is a 25-amino-acid peptide demonstrated to be the iron regulatory hormone capable of blocking iron absorption from the duodenum and iron release from macrophages. Mutations affecting hepcidin regulators or the hepcidin gene itself cause hemochromatosis, a common genetic disorder. Hepcidin is produced mainly by the liver, but many cells and tissues express low levels of the hormone. To determine the contribution of these hepcidin-producing tissues in body iron homeostasis, we have developed a new mouse model in which the hepcidin gene can be conditionally inactivated. Here we compare a liver-specific knockout (KO) mouse model with total KO mice. We show that the liver-specific KO mice fully recapitulate the severe iron overload phenotype observed in the total KO mice, with increased plasma iron and massive parenchymal iron accumulation. This result demonstrates that the hepatocyte constitutes the predominant reservoir for systemic hepcidin and that the other tissues are unable to compensate.
Asunto(s)
Hemocromatosis/genética , Hepcidinas/genética , Hígado/metabolismo , Animales , Modelos Animales de Enfermedad , Marcación de Gen , Hemocromatosis/patología , Hepcidinas/metabolismo , Hierro/metabolismo , Sobrecarga de Hierro/genética , Sobrecarga de Hierro/patología , Hígado/patología , Masculino , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , FenotipoRESUMEN
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 C-terminal region of tubulin is involved in multiple aspects of the regulation of microtubule assembly. To elucidate the molecular mechanisms of this regulation, we study here, using different approaches, the interaction of Tau, spermine, and calcium, three representative partners of the tubulin C-terminal region, with a peptide composed of the last 42 residues of α1a-tubulin. The results show that their binding involves overlapping amino acid stretches in the C-terminal tubulin region: amino acid residues 421-441 for Tau, 430-432 and 444-451 for spermine, and 421-443 for calcium. Isothermal titration calorimetry, NMR, and cosedimentation experiments show that Tau and spermine have similar micromolar binding affinities, whereas their binding stoichiometry differs (C-terminal tubulin peptide/spermine stoichiometry 1:2, and C-terminal tubulin peptide/Tau stoichiometry 8:1). Interestingly, calcium, known as a negative regulator of microtubule assembly, can compete with the binding of Tau and spermine with the C-terminal domain of tubulin and with the positive effect of these two partners on microtubule assembly in vitro. This observation opens up the possibility that calcium may participate in the regulation of microtubule assembly in vivo through direct (still unknown) or indirect mechanism (displacement of microtubule partners). The functional importance of this part of tubulin was also underlined by the observation that an α-tubulin mutant deleted from the last 23 amino acid residues does not incorporate properly into the microtubule network of HeLa cells. Together, these results provide a structural basis for a better understanding of the complex interactions and putative competition of tubulin cationic partners with the C-terminal region of tubulin.
Asunto(s)
Calcio/metabolismo , Espermidina/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas tau/metabolismo , Calcio/química , Cationes/química , Cationes/metabolismo , Células HeLa , Humanos , Microtúbulos/química , Microtúbulos/genética , Microtúbulos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Terciaria de Proteína , Espermidina/química , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Proteínas tau/química , Proteínas tau/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
Upon hypertonic stress most often resulting from high salinity, cells need to balance their osmotic pressure by accumulating neutral osmolytes called compatible osmolytes like betaine, myo-inositol, and taurine. However, the massive uptake of compatible osmolytes is a slow process compared with other defense mechanisms related to oxidative or heat stress. This is especially critical for cycling cells as they have to double their volume while keeping a hospitable intracellular environment for the molecular machineries. Here we propose that clustered cells can accelerate the supply of compatible osmolytes to cycling cells via the transit, mediated by gap junctions, of compatible osmolytes from arrested to cycling cells. Both experimental results in epithelial normal rat kidney cells and theoretical estimations show that gap junctions indeed play a key role in cell adaptation to chronic hypertonicity. These results can provide basis for a better understanding of the functions of gap junctions in osmoregulation not only for the kidney but also for many other epithelia. In addition to this, we suggest that cancer cells that do not communicate via gap junctions poorly cope with hypertonic environments thus explaining the rare occurrence of cancer coming from the kidney medulla.
Asunto(s)
Adaptación Fisiológica/fisiología , Células Epiteliales/fisiología , Uniones Comunicantes/fisiología , Riñón/fisiología , Estrés Fisiológico/fisiología , Equilibrio Hidroelectrolítico/fisiología , Adaptación Fisiológica/efectos de los fármacos , Animales , Betaína/metabolismo , Betaína/farmacología , Comunicación Celular/fisiología , Recuento de Células , Línea Celular , Proliferación Celular/efectos de los fármacos , Conexina 43/genética , Conexina 43/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/patología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/patología , Ácido Glicirretínico/farmacología , Células HeLa , Humanos , Soluciones Hipertónicas , Inositol/metabolismo , Inositol/farmacología , Riñón/citología , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Microtúbulos/patología , Nocodazol/farmacología , Ácidos Oléicos/farmacología , ARN Interferente Pequeño/genética , Ratas , Solución Salina Hipertónica , Fibras de Estrés/metabolismo , Fibras de Estrés/patología , Estrés Fisiológico/efectos de los fármacos , Equilibrio Hidroelectrolítico/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/fisiologíaRESUMEN
Owing to preferential electrostatic adsorption of multivalent cations on highly anionic surfaces, natural multivalent polyamines and especially quadrivalent spermine can be considered as potential regulators of the complex dynamical properties of anionic MTs (microtubules). Indeed, the C-terminal tails of tubulin display many negative residues in a row which should enable the formation of a correlated liquid-like phase of multivalent counterions on its surface. Although it is known that polyamine counterions promote MT assembly in vitro, little is known about the relevance of this interaction in vivo. In the present study, we have explored the relationship between polyamine levels and MT assembly in HeLa and epithelial NRK (normal rat kidney) cells using DFMO (alpha-difluoromethylornithine), an irreversible inhibitor of ornithine decarboxylase, and APCHA [N-(3-aminopropyl)-N-cyclohexylamine], a spermine synthase inhibitor. Under conditions of intracellular polyamine depletion, the MT network is clearly disrupted and the MT mass decreases. Addition of spermine to polyamine-depleted cells reverses this phenotype and rapidly promotes the extensions of the MT network. Finally, we show that polyamine levels modulate the coating of MTs with MAP4 (MT-associated protein 4), an MT-stabilizing protein, and the spatial distribution of EB1 (end-binding protein 1), an MT plus-end-binding protein. In addition, polyamines favour the formation of gap junctions in NRK cells, a process which requires MT extensions at the cell periphery. The present study provides a basis for a better understanding of the role played by polyamines in MT assembly and establishes polyamine metabolism as a potential cellular target for modulating MT functions.
Asunto(s)
Microtúbulos/fisiología , Poliaminas/metabolismo , Animales , Comunicación Celular , Línea Celular , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Uniones Comunicantes/fisiología , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Ratas , Ovinos , Tubulina (Proteína)/fisiologíaRESUMEN
Mutations in SPG4, encoding the microtubule-severing protein spastin, are responsible for the most frequent form of hereditary spastic paraplegia (HSP), a heterogeneous group of genetic diseases characterized by degeneration of the corticospinal tracts. We previously reported that mice harboring a deletion in Spg4, generating a premature stop codon, develop progressive axonal degeneration characterized by focal axonal swellings associated with impaired axonal transport. To further characterize the molecular and cellular mechanisms underlying this mutant phenotype, we have assessed microtubule dynamics and axonal transport in primary cultures of cortical neurons from spastin-mutant mice. We show an early and marked impairment of microtubule dynamics all along the axons of spastin-deficient cortical neurons, which is likely to be responsible for the occurrence of axonal swellings and cargo stalling. Our analysis also reveals that a modulation of microtubule dynamics by microtubule-targeting drugs rescues the mutant phenotype of cortical neurons. Together, these results contribute to a better understanding of the pathogenesis of SPG4-linked HSP and ascertain the influence of microtubule-targeted drugs on the early axonal phenotype in a mouse model of the disease.
Asunto(s)
Adenosina Trifosfatasas/deficiencia , Adenosina Trifosfatasas/genética , Animales , Transporte Axonal , Axones/efectos de los fármacos , Axones/patología , Células Cultivadas , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Ratones , Ratones Noqueados , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Modelos Neurológicos , Mutación , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Nocodazol/farmacología , Paclitaxel/farmacología , Paraplejía Espástica Hereditaria/tratamiento farmacológico , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/metabolismo , Paraplejía Espástica Hereditaria/patología , Espastina , Vinblastina/farmacologíaRESUMEN
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
Clostridium botulinum exoenzyme C3 is responsible for the inactivation of members of the Rho GTPase family that are implicated in actin-cytoskeleton reorganization. This property has been extensively used in the field to investigate the functionality of the Rho GTPases. However, systematic analysis of Rho GTPase functions requires large amounts of such inhibitors and consequently an optimization of the production yield of these proteins. Bacterial production of soluble proteins often requires a refolding step that noticeably affects the production yields and necessitates additional experiments to verify functional activity. This is particularly true for TAT-C3, the production yields of which are generally low. In this report, we describe a rapid and efficient method for the production of soluble C3 exoenzyme developed by screening a collection of bacterial strains. The recombinant C3 protein was fused to the TAT protein-transduction domain from HIV, to allow protein delivery into cells, and to a hexahistidine tag, that permitted purification by Nickel affinity chromatography. We have demonstrated the production of large amounts of soluble and functional protein using the bacterial strain AD494 (DE3)pLysS. This rapid and efficient method for the production of soluble C3 exoenzyme could also be useful for the production of other proteins with solubility problems.