RESUMEN
The prolyl-4-hydroxylase domain 1-3 (PHD1-3) enzymes are regulating the protein stability of the α-subunit of the hypoxia-inducible factor-1 (HIF-1), which mediates oxygen-dependent gene expression. PHD2 is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. In human cancers, HIF-1α is overexpressed as a result of intratumoral hypoxia which in turn promotes tumor progression. The role of PHD2 for tumor progression is in contrast far from being thoroughly understood. Therefore, we established PHD2 knockdown clones of MDA-MB-231 breast cancer cells and analyzed their tumor-forming potential in a SCID mouse model. Tumor progression was significantly impaired in the PHD2 knockdown MDA-MB-231 cells, which could be partially rescued by re-establishing PHD2 expression. In a RNA profile screen, we identified the secreted phosphoprotein 1 (SPP1) as one target, which is differentially regulated as a consequence of the PHD2 knockdown. Knockdown of PHD2 drastically reduced the SPP1 expression in MDA-MB-231 cells. A correlation of SPP1 and PHD2 expression was additionally verified in 294 invasive breast cancer biopsies. In subsequent analyses, we identified that PHD2 alters the processing of transforming growth factor (TGF)-ß1, which is highly involved in SPP1 expression. The altered processing capacity was associated with a dislocation of the pro-protein convertase furin. Thus, our data demonstrate that in MDA-MB-231 cells PHD2 might affect tumor-relevant TGF-ß1 target gene expression by altering the TGF-ß1 processing capacity.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Neoplasias de la Mama/patología , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Ratones , Osteopontina/genética , Transducción de Señal , Carga Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cells are responding to hypoxia via prolyl-4-hydroxylase domain (PHD) enzymes, which are responsible for oxygen-dependent hydroxylation of the hypoxia-inducible factor (HIF)-1α subunit. To gain further insight into PHD function, we generated knockdown cell models for the PHD2 isoform, which is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. Induction of a PHD2 knockdown in tetracycline-inducible HeLa PHD2 knockdown cells resulted in increased F-actin formation as detected by phalloidin staining. A similar effect could be observed in the stably transfected PHD2 knockdown cell clones 1B6 and 3B7. F-actin is at least in part responsible for shaping cell morphology as well as regulating cell migration. Cell migration was impaired significantly as a consequence of PHD2 knockdown in a scratch assay. Mechanistically, PHD2 knockdown resulted in activation of the RhoA (Ras homolog gene family member A)/Rho-associated kinase pathway with subsequent phosphorylation of cofilin. Because cofilin phosphorylation impairs its actin-severing function, this may explain the F-actin phenotype, thereby providing a functional link between PHD2-dependent signaling and cell motility.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Movimiento Celular , Citoesqueleto/metabolismo , Células HeLa , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Modelos Biológicos , Fosforilación , Polímeros/química , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de ProteínaRESUMEN
Polystyrene nanoparticles (PS-NPs) were doped with an oxygen-sensitive near-infrared (NIR)-emissive palladium meso-tetraphenylporphyrin and an inert reference dye which are both excitable at 635 nm. The nanosensors were characterized with special emphasis on fundamental parameters such as absolute photoluminescence quantum yield and fluorescence lifetime. The PS-NPs were employed for ratiometric dual-wavelength and lifetime-based photoluminescent oxygen sensing. They were efficiently taken up by cultured murine alveolar macrophages, yielding a characteristic and reversible change in ratiometric response with decreasing oxygen concentration. This correlated with the cellular hypoxic status verified by analysis of hypoxia inducible factor-1α (HIF-1α) accumulation. In addition, the surface of PS-NPs was functionalized with polyethylene glycol (PEG) and the monoclonal antibody herceptin, and their binding to HER2/neu-overexpressing tumor cells was confirmed in vitro. First experiments with tumor-bearing mouse revealed a distinctive ratiometric response within the tumor upon hypoxic condition induced by animal sacrifice. These results demonstrate the potential of these referenced NIR nanosensors for in vitro and in vivo imaging that present a new generation of optical probes for oncology.
Asunto(s)
Hipoxia , Nanopartículas/química , Neoplasias/diagnóstico , Poliestirenos/química , Espectroscopía Infrarroja Corta , Animales , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/metabolismo , Línea Celular Tumoral , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Ratones , Paladio/química , Polietilenglicoles/química , Porfirinas/química , Unión Proteica , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , TrastuzumabRESUMEN
In Fusarium fujikuroi, the production of gibberellins and bikaverin is repressed by nitrogen sources such as glutamine or ammonium. Sensing and uptake of ammonium by specific permeases play key roles in nitrogen metabolism. Here, we describe the cloning of three ammonium permease genes, mepA, mepB, and mepC, and their participation in ammonium uptake and signal transduction in F. fujikuroi. The expression of all three genes is strictly regulated by the nitrogen regulator AreA. Severe growth defects of DeltamepB mutants on low-ammonium medium and methylamine uptake studies suggest that MepB functions as the main ammonium permease in F. fujikuroi. In DeltamepB mutants, nitrogen-regulated genes such as the gibberellin and bikaverin biosynthetic genes are derepressed in spite of high extracellular ammonium concentrations. mepA mepB and mepC mepB double mutants show a similar phenotype as DeltamepB mutants. All three F. fujikuroi mep genes fully complemented the Saccharomyces cerevisiae mep1 mep2 mep3 triple mutant to restore growth on low-ammonium medium, whereas only MepA and MepC restored pseudohyphal growth in the mep2/mep2 mutant. Overexpression of mepC in the DeltamepB mutants partially suppressed the growth defect but did not prevent derepression of AreA-regulated genes. These studies provide evidence that MepB functions as a regulatory element in a nitrogen sensing system in F. fujikuroi yet does not provide the sensor activity of Mep2 in yeast, indicating differences in the mechanisms by which nitrogen is sensed in S. cerevisiae and F. fujikuroi.
Asunto(s)
Proteínas Fúngicas/fisiología , Fusarium/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Proteínas de Transporte de Membrana/metabolismo , Nitrógeno/farmacología , Compuestos de Amonio Cuaternario/metabolismo , Northern Blotting , Southern Blotting , Clonación Molecular , Proteínas Fúngicas/genética , Fusarium/crecimiento & desarrollo , Fusarium/patogenicidad , Eliminación de Gen , Prueba de Complementación Genética , Proteínas de Transporte de Membrana/genética , Filogenia , Reacción en Cadena de la Polimerasa , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Transformación GenéticaRESUMEN
Clinical studies in breast cancer suggest important associations between intratumoral hypoxia, the upregulation of epidermal growth factor receptor (EGFR or HER1), hypoxia-inducible factor 1α (HIF-1α), and reduced patient survival. However, direct molecular links between EGFR and the hypoxia signaling system are not yet established. Since the oxygen sensor hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) is considered to be the main HIF-1α regulator, we hypothesized that PHD2 and EGFR may be interconnected at the molecular level. By analyzing samples from 313 breast cancer patients, we found that EGFR is a first clinicopathological parameter positively correlating with PHD2. Mechanistically, we identified PHD2 as a direct binding partner of EGFR and show that PHD2 regulates EGFR stability as well as its subsequent signaling in breast carcinoma cells. Overall, we introduce for the first time the direct crosstalk between the oxygen sensor PHD2 and EGFR-mediated tumorigenesis in breast cancer.
Asunto(s)
Neoplasias de la Mama/enzimología , Receptores ErbB/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Transducción de Señal , Hipoxia Tumoral , Microambiente Tumoral , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/agonistas , Receptores ErbB/genética , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Fosforilación , Unión Proteica , Estabilidad Proteica , Proteolisis , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , TransfecciónRESUMEN
On a molecular level, cells sense changes in oxygen availability through the PHDs, which regulate the protein stability of the α-subunit of the transcription factor HIF. Especially, PHD3 has been additionally associated with apoptotic cell death. We hypothesized that PHD3 plays a role in cell-fate decisions in macrophages. Therefore, myeloid-specific PHD3(-/-) mice were created and analyzed. PHD3(-/-) BMDM showed no altered HIF-1α or HIF-2α stabilization or increased HIF target gene expression in normoxia or hypoxia. Macrophage M1 and M2 polarization was unchanged likewise. Compared with macrophages from WT littermates, PHD3(-/-) BMDM exhibited a significant reduction in TUNEL-positive cells after serum withdrawal or treatment with stauro and SNAP. Under the same conditions, PHD3(-/-) BMDM also showed less Annexin V staining, which is representative for membrane disruption, and indicated a reduced early apoptosis. In an unbiased transcriptome screen, we found that Angptl2 expression was reduced in PHD3(-/-) BMDM under stress conditions. Addition of rAngptl2 rescued the antiapoptotic phenotype, demonstrating that it is involved in the PHD3-mediated response toward apoptotic stimuli in macrophages.
Asunto(s)
Macrófagos/citología , Procolágeno-Prolina Dioxigenasa/fisiología , Proteína 2 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Angiopoyetinas/biosíntesis , Angiopoyetinas/genética , Angiopoyetinas/farmacología , Animales , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Células de la Médula Ósea/citología , Hipoxia de la Célula , Células Cultivadas , Regulación de la Expresión Génica , Hidroxilación , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/enzimología , FN-kappa B/metabolismo , Procolágeno-Prolina Dioxigenasa/deficiencia , Procolágeno-Prolina Dioxigenasa/genética , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/farmacología , S-Nitroso-N-Acetilpenicilamina/farmacología , Estaurosporina/farmacología , Transcripción Genética , TranscriptomaRESUMEN
The activating transcription factor-4 (ATF-4) is translationally induced under anoxic conditions, mediates part of the unfolded protein response following endoplasmic reticulum (ER) stress, and is a critical regulator of cell fate. Here, we identified the zipper II domain of ATF-4 to interact with the oxygen sensor prolyl-4-hydroxylase domain 3 (PHD3). The PHD inhibitors dimethyloxalylglycine (DMOG) and hypoxia, or proteasomal inhibition, all induced ATF-4 protein levels. Hypoxic induction of ATF-4 was due to increased protein stability, but was independent of the ubiquitin ligase von Hippel-Lindau protein (pVHL). A novel oxygen-dependent degradation (ODD) domain was identified adjacent to the zipper II domain. Mutations of 5 prolyl residues within this ODD domain or siRNA-mediated down-regulation of PHD3, but not of PHD2, was sufficient to stabilize ATF-4 under normoxic conditions. These data demonstrate that PHD-dependent oxygen-sensing recruits both the hypoxia-inducible factor (HIF) and ATF-4 systems, and hence not only confers adaptive responses but also cell fate decisions.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Dioxigenasas/fisiología , Oxígeno/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Factor de Transcripción Activador 4/química , Secuencia de Aminoácidos , Hipoxia de la Célula/fisiología , Dioxigenasas/química , Células HeLa , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
In Fusarium fujikuroi, the biosynthesis of gibberellins (GAs) and bikaverin is under control of AreA-mediated nitrogen metabolite repression. Thus far, the signaling components acting upstream of AreA and regulating its nuclear translocation are unknown. In Saccharomyces cerevisiae, the target of rapamycin (TOR) proteins, Tor1p and Tor2p, are key players of nutrient-mediated signal transduction to control cell growth. In filamentous fungi, probably only one TOR kinase-encoding gene exists. However, nothing is known about its function. Therefore, we investigated the role of TOR in the GA-producing fungus F. fujikuroi in order to determine whether TOR plays a role in nitrogen regulation, especially in the regulation of GA and bikaverin biosynthesis. We cloned and characterized the F. fujikuroi tor gene. However, we were not able to create knockout mutants, suggesting that TOR is essential for viability. Inhibition of TOR by rapamycin affected the expression of AreA-controlled secondary metabolite genes for GA and bikaverin biosynthesis, as well as genes involved in transcriptional and translational regulation, ribosome biogenesis, and autophagy. Deletion of fpr1 encoding the FKBP12-homologue confirmed that the effects of rapamycin are due to the specific inhibition of TOR. Interestingly, the expression of most of the TOR target genes has been previously shown to be also affected in the glutamine synthetase mutant, although in the opposite way. We demonstrate here for the first time in a filamentous fungus that the TOR kinase is involved in nitrogen regulation of secondary metabolism and that rapamycin affects also the expression of genes involved in translation control, ribosome biogenesis, carbon metabolism, and autophagy.