RESUMEN
During acute viral infections, innate immune cells invade inflamed tissues and face hypoxic areas. Hypoxia-inducible factors (HIFs) adapt cellular responses towards these conditions. We wanted to investigate the effects of a loss of HIF-2α in macrophages during acute Friend murine leukemia retrovirus (FV) infection in C57BL/6 mice using a Cre/loxP system. Remarkably, mice with floxed Hif-2a (Hif-2afl; Hif-2a is also known as Epas1) did not show any signs of FV infection independent of Cre activity. This prevented a detailed analysis of the role of macrophage HIF-2α for FV infection but allowed us to study a model of unexpected FV resistance. Hif-2afl mice showed a significant decrease in the expression of the Atp6v1e2 gene encoding for the E2 subunit of the vacuolar H+-ATPase, which resulted in a decreased acidification of lysosomes and limited virus entry into the cell. These findings highlight that the insertion of loxP sites is not always without functional consequences and has established a phenotype in the floxed Hif-2a mouse, which is not only unexpected, but unwanted and is of relevance for the use of this mouse strain in (at least virus) experiments.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Virus de la Leucemia Murina de Friend , ATPasas de Translocación de Protón Vacuolares , Animales , Ratones , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Virus de la Leucemia Murina de Friend/genética , Lisosomas/metabolismo , Macrófagos/metabolismo , Macrófagos/virología , Macrófagos/inmunología , Ratones Endogámicos C57BL , Infecciones por Retroviridae/genética , Infecciones por Retroviridae/metabolismo , Infecciones por Retroviridae/virología , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
Replication protein A (RPA) is the major single-stranded DNA (ssDNA) binding protein that is essential for DNA replication and processing of DNA double-strand breaks (DSBs) by homology-directed repair pathways. Recently, small molecule inhibitors have been developed targeting the RPA70 subunit and preventing RPA interactions with ssDNA and various DNA repair proteins. The rationale of this development is the potential utility of such compounds as cancer therapeutics, owing to their ability to inhibit DNA replication that sustains tumor growth. Among these compounds, (1Z)-1-[(2-hydroxyanilino) methylidene] naphthalen-2-one (HAMNO) has been more extensively studied and its efficacy against tumor growth was shown to arise from the associated DNA replication stress. Here, we study the effects of HAMNO on cells exposed to ionizing radiation (IR), focusing on the effects on the DNA damage response and the processing of DSBs and explore its potential as a radiosensitizer. We show that HAMNO by itself slows down the progression of cells through the cell cycle by dramatically decreasing DNA synthesis. Notably, HAMNO also attenuates the progression of G2-phase cells into mitosis by a mechanism that remains to be elucidated. Furthermore, HAMNO increases the fraction of chromatin-bound RPA in S-phase but not in G2-phase cells and suppresses DSB repair by homologous recombination. Despite these marked effects on the cell cycle and the DNA damage response, radiosensitization could neither be detected in exponentially growing cultures, nor in cultures enriched in G2-phase cells. Our results complement existing data on RPA inhibitors, specifically HAMNO, and suggest that their antitumor activity by replication stress induction may not extend to radiosensitization. However, it may render cells more vulnerable to other forms of DNA damaging agents through synthetically lethal interactions, which requires further investigation.
Asunto(s)
Neoplasias , Proteína de Replicación A , Humanos , Proteína de Replicación A/metabolismo , Ciclo Celular/genética , Proteínas de Unión al ADN/metabolismo , Replicación del ADN , Reparación del ADN , Daño del ADN , ADN , Mitosis , ADN de Cadena SimpleRESUMEN
Carotid body (CB) type I cells sense the blood Po2 and generate a nervous signal for stimulating ventilation and circulation when blood oxygen levels decline. Three oxygen-sensing enzyme complexes may be used for this purpose: 1) mitochondrial electron transport chain metabolism, 2) heme oxygenase 2 (HO-2)-generating CO, and/or 3) an NAD(P)H oxidase (NOX). We hypothesize that intracellular redox changes are the link between the sensor and nervous signals. To test this hypothesis type I cell autofluorescence of flavoproteins (Fp) and NAD(P)H within the mouse CB ex vivo was recorded as Fp/(Fp+NAD(P)H) redox ratio. CB type I cell redox ratio transiently declined with the onset of hypoxia. Upon reoxygenation, CB type I cells showed a significantly increased redox ratio. As a control organ, the non-oxygen-sensing sympathetic superior cervical ganglion (SCG) showed a continuously reduced redox ratio upon hypoxia. CN-, diphenyleneiodonium, or reactive oxygen species influenced chemoreceptor discharge (CND) with subsequent loss of O2 sensitivity and inhibited hypoxic Fp reduction only in the CB but not in SCG Fp, indicating a specific role of Fp in the oxygen-sensing process. Hypoxia-induced changes in CB type I cell redox ratio affected peptidyl prolyl isomerase Pin1, which is believed to colocalize with the NADPH oxidase subunit p47phox in the cell membrane to trigger the opening of potassium channels. We postulate that hypoxia-induced changes in the Fp-mediated redox ratio of the CB regulate the Pin1/p47phox tandem to alter type I cell potassium channels and therewith CND.
Asunto(s)
Cuerpo Carotídeo/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Oxígeno/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células Quimiorreceptoras/metabolismo , Flavoproteínas/metabolismo , Hipoxia/metabolismo , Pulmón/metabolismo , Ratones , Mitocondrias/metabolismo , Canales de Potasio/metabolismoRESUMEN
Autophagy is commonly described as a cell survival mechanism and has been implicated in chemo- and radioresistance of cancer cells. Whether ionizing radiation induced autophagy triggers tumor cell survival or cell death still remains unclear. In this study the autophagy related proteins Beclin1 and ATG7 were tested as potential targets to sensitize colorectal carcinoma cells to ionizing radiation under normoxic, hypoxic and starvation conditions. Colony formation, apoptosis and cell cycle analysis revealed that knockdown of Beclin1 or ATG7 does not enhance radiosensitivity in HCT-116 cells. Furthermore, ATG7 knockdown led to an increased survival fraction under oxygen and glutamine starvation, indicating that ionizing radiation indeed induces autophagy which, however, leads to cell death finally. These results highlight that inhibition of autophagic pathways does not generally increase therapy success but may also lead to an unfavorable outcome especially under amino acid and oxygen restriction.
Asunto(s)
Apoptosis/efectos de la radiación , Autofagia/efectos de la radiación , Neoplasias Colorrectales/patología , Radiación Ionizante , Proteína 7 Relacionada con la Autofagia/metabolismo , Beclina-1/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Glutamina/deficiencia , Humanos , Oxígeno/farmacologíaRESUMEN
BACKGROUND: The nudix family member enzyme MutT homologue-1 (MTH1) hydrolyses the oxidized nucleotides 8-oxo-dGTP and 2-hydroxy-dATP and thus prevents the incorporation of damaged nucleotides into nuclear and mitochondrial DNA. Therefore MTH1 was proposed to protect cancer cells from oxidative DNA lesions and subsequent cell death. We investigated whether the bona fide MTH1 inhibitor TH588 affects responses of cultured colorectal tumor cells to ionizing radiation (IR) in normoxia and in moderate or severe hypoxia. METHODS: TH588 was tested in cell viability and survival assays (tetrazolium dye (MTT), propidium iodide staining, caspase-3 activity, and colony formation assays (CFA)) in colorectal carcinoma cells (HCT116 and SW480) in combination with IR in normoxia and in hypoxia. Additionally, MTH1 was targeted by lentiviral shRNA expression. Human umbilical vein endothelial cells (HUVEC) were assessed in MTT assays. RESULTS: In all cell lines tested, TH588 dose-dependently impaired cell survival. In CFAs, TH588 and IR effects on carcinoma cells were additive in normoxia and in hypoxia. Using 3 different shRNAs, the lentiviral approach was detrimental to SW480, but not to HCT116. CONCLUSIONS: TH588 has cytotoxic effects on transformed and untransformed cells and synergizes with IR in normoxia and in hypoxia. TH588 toxicity is not fully explained by MTH1 inhibition as HCT116 were unaffected by lentiviral suppression of MTH1 expression. TH588 should be explored further because it has radiosensitizing effects in hypoxia.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Hipoxia/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Pirimidinas/farmacología , Tolerancia a Radiación/efectos de los fármacos , Radiación Ionizante , Fármacos Sensibilizantes a Radiaciones/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/radioterapia , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Estrés Oxidativo/efectos de los fármacosRESUMEN
The asparaginyl hydroxylase factor inhibiting HIF-1 (FIH-1) is an important suppressor of hypoxia-inducible factor (HIF) activity. In addition to HIF-α, FIH-1 was previously shown to hydroxylate other substrates within a highly conserved protein interaction domain, termed the ankyrin repeat domain (ARD). However, to date, the biological role of FIH-1-dependent ARD hydroxylation could not be clarified for any ARD-containing substrate. The apoptosis-stimulating p53-binding protein (ASPP) family members were initially identified as highly conserved regulators of the tumour suppressor p53. In addition, ASPP2 was shown to be important for the regulation of cell polarity through interaction with partitioning defective 3 homolog (Par-3). Using mass spectrometry we identified ASPP2 as a new substrate of FIH-1 but inhibitory ASPP (iASPP) was not hydroxylated. We demonstrated that ASPP2 asparagine 986 (N986) is a single hydroxylation site located within the ARD. ASPP2 protein levels and stability were not affected by depletion or inhibition of FIH-1. However, FIH-1 depletion did lead to impaired binding of Par-3 to ASPP2 while the interaction between ASPP2 and p53, apoptosis and proliferation of the cancer cells were not affected. Depletion of FIH-1 and incubation with the hydroxylase inhibitor dimethyloxalylglycine (DMOG) resulted in relocation of ASPP2 from cell-cell contacts to the cytosol. Our data thus demonstrate that protein interactions of ARD-containing substrates can be modified by FIH-1-dependent hydroxylation. The large cellular pool of ARD-containing proteins suggests that FIH-1 can affect a broad range of cellular functions and signalling pathways under certain conditions, for example, in response to severe hypoxia.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Oxigenasas de Función Mixta/metabolismo , Proteínas Represoras/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Adhesión Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Procesos de Crecimiento Celular/fisiología , Hipoxia de la Célula/fisiología , Línea Celular , Línea Celular Tumoral , Movimiento Celular/fisiología , Polaridad Celular/fisiología , Citosol/metabolismo , Citosol/fisiología , Células HCT116 , Células HEK293 , Humanos , Hidroxilación , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Mapas de Interacción de Proteínas , Estructura Terciaria de Proteína/fisiología , Alineación de Secuencia , Transducción de SeñalRESUMEN
Reactive oxygen species (ROS) generated by the NADPH oxidase have been proposed to play an important role in the carotid body (CB) oxygen sensing process (Cross et al. 1990). Up to now it remains unclear whether hypoxia causes an increase or decrease of CB ROS levels. We transfected CBs with the ROS sensitive HSP-FRET construct and subsequently measured the intracellular redox state by means of Förster resonance energy transfer (FRET) microscopy. In a previous study we found both increasing and decreasing ROS levels under hypoxic conditions. The transition from decreasing to increasing ROS levels coincided with the change of the caging system from ambient environment caging (AEC) to individually ventilated caging (IVC) (Bernardini A, Brockmeier U, Metzen E, Berchner-Pfannschmidt U, Harde E, Acker-Palmer A, Papkovsky D, Acker H, Fandrey J, Type I cell ROS kinetics under hypoxia in the intact mouse carotid body ex vivo: a FRET based study. Am J Physiol Cell Physiol. doi: 10.1152/ajpcell.00370.2013 , 2014). In this work we analyze hypoxia induced ROS reaction of animals from an IVC system that had been exposed to AEC conditions for 5 days. The results further support the hypothesis of an important impact of the caging system on CB ROS reaction.
Asunto(s)
Cuerpo Carotídeo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Animales , Transferencia Resonante de Energía de Fluorescencia , Potenciales de la MembranaRESUMEN
BACKGROUND: Pancreatic cancer is 1 of the most common and poorly treated tumors. In search of new therapeutic approaches, the oxygen sensors prolyl hydroxylases (PHD) are potential targets. PHD2 is considered the key oxygen sensor-regulating hypoxia-inducible factor (HIF). Currently, there is conflicting evidence regarding the exact role of PHD2 in tumorigenesis. The objective of this study was to investigate the role of PHD2 in pancreatic cancer growth and progression. METHODS: PHD2 expression was analyzed by quantitative real-time polymerase chain reaction analysis and immunohistochemistry in human tissue specimens and cell lines. Knockdown of PHD2 was done by using short-interfering RNAs (siRNAs) specific against PHD2, and PHD2 overexpression was achieved by stable combinational DNA transfection. In vivo, an orthotopic murine model was used. Angiogenic cytokines were assessed with enzyme-linked immunosorbent assays, and invasion was studied with Matrigel assays. RESULTS: PHD2 expression was not altered substantially in cancer tissues and their metastases. Lymph node-negative tissues had higher levels of PHD2 than lymph node-positive tissues. PHD2 was hypoxia-inducible in pancreatic cancer cell lines and regulated cell growth through cyclin D1 down-regulation samples with PHD2 suppression and through p21 up-regulation in samples with of PHD2 overexpression. In vivo, PHD2 caused tumor growth retardation and reduced tumor invasion by inhibiting angiogenesis. This observation was caused by the suppression of angiogenic cytokines and tumor invasion. CONCLUSIONS: The current results indicated that PHD2 plays an important role in pancreatic tumorigenesis. In summary, the authors concluded that PHD2 may function as a tumor suppressor gene in pancreatic cancer and, thus, may define a potential target for the treatment of pancreatic cancer.
Asunto(s)
Proliferación Celular , Neovascularización Patológica/fisiopatología , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/fisiopatología , Procolágeno-Prolina Dioxigenasa/fisiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Estudios de Casos y Controles , Línea Celular Tumoral , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Hipoxia/fisiopatología , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica/fisiopatología , Neoplasias Pancreáticas/irrigación sanguínea , Estudios Retrospectivos , Trasplante Heterólogo , Adulto JovenRESUMEN
The transcription factor hypoxia-inducible factor (HIF) is the main oxygen sensor which regulates adaptation to cellular hypoxia. The aim of this study was to establish cultured murine hepatocyte derived cells (mHDC) as an in vitro model and to analyze the role of HIF-1α in apoptosis induction, DNA damage repair and sensitivity to ionizing radiation (IR). We have crossed C57/BL6 mice that bear loxP sites flanking exon 2 of Hif1a with mice which carry tamoxifen-inducible global Cre expression. From the offspring, we have established transduced hepatocyte cultures which are permanently HIF-1α deficient after tamoxifen treatment. We demonstrated that the cells produce albumin, acetylcholine esterase, and the cytokeratins 8 and 18 which functionally characterizes them as hepatocytes. In moderate hypoxia, HIF-1α deficiency increased IR-induced apoptosis and significantly reduced the surviving fraction of mHDC as compared to HIF-1α expressing cells in colony formation assays. Furthermore, HIF-1α knockout cells displayed increased IR-induced DNA damage as demonstrated by increased generation and persistence of γH2AX foci. HIF-1α deficient cells showed delayed DNA repair after IR in hypoxia in neutral comet assays which may indicate that non-homologous end joining (NHEJ) repair capacity was affected. Overall, our data suggest that HIF-1α inactivation increases radiation sensitivity of mHDC cells.
Asunto(s)
Hepatocitos , Hipoxia , Animales , Hepatocitos/metabolismo , Hipoxia/metabolismo , Integrasas , Ratones , Radiación Ionizante , TamoxifenoRESUMEN
BACKGROUND: Radiotherapy is one of the most widely used treatments for cancer. The benefit of radiation is known to be negatively affected by tumor hypoxia and the expression of hypoxia-inducible factors (HIF), respectively. HIF-1α/ ß and HIF-2α/ ß are transcriptional activators of oxygen-regulated genes. The aim of the study was to examine cell type-specific effects of HIF-1α and -2α knockdown or oxygen-independent HIF-stabilisation on radiosensitivity. METHODS: Herein, we treated four different wildtype and HIF-1α- or HIF-2α-deficient human cancer cell lines, cultured under normoxic or hypoxic conditions, with ionising radiation in doses from 2 to 6 Gy and examined clonogenic survival. Furthermore, the cells were partly preincubated with a HIF-stabiliser (di-tert-butyroyl-oxymethyl-2,4-pyridine-dicarboxylate, (t)Bu-2,4-PDC). RESULTS: The results show that both hypoxia exposure and treatment with (t)Bu-2,4-PDC increased the radioresistance of human cancer cells. The HIF-mediated decrease of radioresponsiveness induced by the chemical stabiliser emerged to be as strong as the one caused by hypoxia. Clonogenic survival assays furthermore revealed that HIF-1 expression enhanced resistance to radiation, whereas knocking-down HIF-1 increased the sensitivity to radiation under normoxic as well as under hypoxic conditions. CONCLUSION: These data extend previous observations of HIF-1α and broaden the view by showing HIF-2α inverse correlation between HIF expression and prognosis for the outcome of radiotherapy.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Rayos gamma , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Hipoxia de la Célula , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias/radioterapia , Ácidos Pipecólicos/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
The disulfide isomerase ERp57, originally found in the endoplasmic reticulum, is located in multiple cellular compartments, participates in diverse cell functions and interacts with a huge network of binding partners. It was recently suggested as an attractive new target for cancer therapy due to its critical role in tumor cell proliferation. Since a major bottleneck in cancer treatment is the occurrence of hypoxic areas in solid tumors, the role of ERp57 in cell growth was tested under oxygen depletion in the colorectal cancer cell line HCT116. We observed a severe growth inhibition when ERp57 was knocked down in hypoxia (1% O2) as a consequence of downregulated c-Myc, PLK1, PDPK1 (PDK1) and AKT (PKB). Further, irradiation experiments revealed also a radiosensitizing effect of ERp57 depletion under oxygen deprivation. Compared to ERp57, we do not favour PDPK1 as a suitable pharmaceutical target as its efficient knockdown/chemical inhibition did not show an inhibitory effect on proliferation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Neoplasias del Colon/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Hipoxia Tumoral , Apoptosis , Proliferación Celular , Neoplasias del Colon/genética , Neoplasias del Colon/radioterapia , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HCT116 , Humanos , Oxígeno/metabolismo , Proteína Disulfuro Isomerasas/genética , Transducción de Señal , Quinasa Tipo Polo 1RESUMEN
Hypoxia-induced resistance of tumor cells to therapeutic treatment is an unresolved limitation due to poor vascular accessibility and protective cell adaptations provided by a network, including PERK, NRF2, and HIF signaling. All three pathways have been shown to influence each other, but a detailed picture remains elusive. To explore this crosstalk in the context of tumor therapy, we generated human cancer cell lines of pancreatic and lung origin carrying an inducible shRNA against NRF2 and PERK. We report that PERK-related phosphorylation of NRF2 is only critical in Keap1 wildtype cells to escape its degradation, but shows no direct effect on nuclear import or transcriptional activity of NRF2. We could further show that NRF2 is paramount for proliferation, ROS elimination, and radioprotection under constant hypoxia (1% O2), but is dispensable under normoxic conditions or after reoxygenation. Depletion of NRF2 does not affect apoptosis, cell cycle progression and proliferation factors AKT and c-Myc, but eliminates cellular HIF-1α signaling. Co-IP experiments revealed a protein interaction between NRF2 and HIF-1α and strongly suggest NRF2 as one of the cellular key factor for the HIF pathway. Together these data provide new insights on the complex role of the PERK-NRF2-HIF-axis for cancer growth.
Asunto(s)
Hipoxia de la Célula/genética , Neoplasias Pulmonares/genética , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias Pancreáticas/genética , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pancreáticas/patología , Transducción de Señal , TransfecciónRESUMEN
Upon ER stress cells activate the unfolded protein response through PERK, IRE1 and ATF6. Remarkable effort has been made to delineate the downstream signaling of these three ER stress sensors after activation, but upstream regulation at the ER luminal site still remains mostly undefined. Here we report that the thiol oxidoreductase PDI is mandatory for activation of the PERK pathway in HEK293T as well as in human pancreatic, lung and colon cancer cells. Under ER stress, depletion of PDI selectively abrogated eIF2α phosphorylation, induction of ATF4, CHOP and even BiP. Furthermore, we could demonstrate that PDI prevented degradation of activated PERK by the 26S proteasome and therefore contributes to maintained PERK signaling. As a result of decreased PERK activity, PDI depleted cells showed an increased vulnerability to ER stress induced by chemicals or ionizing radiation in 2D as well as in 3D culture models. We conclude that PDI is an obligatory regulator of the PERK pathway with future therapy implications.
Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Oxidorreductasas/metabolismo , Transducción de Señal/fisiología , eIF-2 Quinasa/metabolismo , Células A549 , Apoptosis/fisiología , Línea Celular , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Células HCT116 , Células HEK293 , Humanos , Neoplasias/metabolismo , Fosforilación/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Disulfuro Isomerasas/metabolismoRESUMEN
Hypoxia-inducible factors are the key elements in the essential process of oxygen homeostasis of vertebrate cells. Stabilisation and subsequent nuclear localisation of HIF-alpha subunits results in the activation of target genes such as vegf, epo and glut1. The passage of transcription factors e.g. HIF-1alpha into the nucleus through the nuclear pore complex is regulated by nuclear transport receptors. Therefore nucleocytoplasmic shuttling can regulate transcriptional activity by facilitating the cellular traffic of transcription factors between both compartments. Here, we report on the identification of specific interactions of hypoxia-inducible factors with nuclear transport receptors importin alpha/beta. HIF-1alpha, -1beta, and HIF-2alpha are binding to importin alpha1, alpha3, alpha5, and alpha7. The direct interaction of HIF-1alpha to alpha importins is dependent on a functional nuclear localisation signal within the C-terminal region of the protein. In contrast, the supposed N-terminal NLS is not effective. Our findings provide new insight into the mechanism of the regulation of nuclear transport of hypoxia-inducible factors.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Núcleo Celular/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , alfa Carioferinas/fisiología , beta Carioferinas/fisiología , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Sitios de Unión , Células Cultivadas , Células HeLa , Humanos , Señales de Localización Nuclear/química , Señales de Localización Nuclear/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/metabolismo , Transducción de SeñalRESUMEN
Erythropoietin (Epo) therapy reduces red cell transfusion requirements and improves the quality of life of anemic cancer patients receiving chemotherapy. However, there is concern that Epo may promote tumor growth. We investigated by real-time RT-PCR, immunofluorescence microscopy, Western blotting and cell growth analysis whether human cancer cell lines (SH-SY5Y, MCF7, HepG2, U2-OS, HeLa, HEK293T, RCC4, HCT116, 7860wt and SW480) possess functional Epo receptors (EpoR). We detected EpoR mRNA in all cell lines. Neither hypoxia nor Epo treatment altered the level of EpoR mRNA expression. Four commonly used commercial antibodies proved to be unsuitable for immunoblot procedures because they cross-reacted with several proteins unrelated with EpoR. Depending on the antibody used, EpoR was localized to the plasma membrane, the cytoplasm or the nucleus. Experiments with small interfering RNA showed that EpoR protein was not expressed by the tumor cells except by UT7/Epo leukemia cells, which served as an EpoR positive control line, and by cells transfected with the human EpoR gene. Apart from UT7/Epo, none of the tumor cell lines responded to Epo treatment with phosphorylation of signaling molecules or with cell proliferation.
Asunto(s)
Eritropoyetina/farmacología , Neoplasias/metabolismo , Receptores de Eritropoyetina/biosíntesis , Transducción de Señal/efectos de los fármacos , Anticuerpos/inmunología , Western Blotting , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Proliferación Celular , Reacciones Cruzadas , Técnica del Anticuerpo Fluorescente , Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Fosforilación , ARN Mensajero/análisis , Receptores de Eritropoyetina/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Hypoxia-inducible factor-1 (HIF-1) coordinates the cellular response to a lack of oxygen by controlling the expression of hypoxia-inducible genes that ensure an adequate energy supply. Assembly of the HIF-1 complex by its oxygen-regulated subunit HIF-1alpha and its constitutive beta subunit also known as ARNT is the key event of the cellular genetic response to hypoxia. By two-photon microscopy, we studied HIF-1 assembly in living cells and the mobility of fluorophore-labeled HIF-1 subunits by fluorescence recovery after photobleaching. We found a significantly slower nuclear migration of HIF-1alpha than of HIF-1beta, indicating that each subunit can move independently. We applied fluorescence resonance energy transfer to calculate the nanometer distance between alpha and beta subunits of the transcriptionally active HIF-1 complex bound to DNA. Both N termini of the fluorophore-labeled HIF-1 subunits were localized as close as 6.2 nm, but even the N and C terminus of the HIF-1 complex were not further apart than 7.4 nm. Our data suggest a more compact 3-dimensional organization of the HIF complex than described so far by 2-dimensional models.
Asunto(s)
Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Subunidad alfa del Factor 1 Inducible por Hipoxia/análisis , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Subunidades de Proteína/análisis , Células Tumorales CultivadasRESUMEN
The transcriptional activator HIF (hypoxia-inducible factor) is a focal point of biomedical research because many situations in physiology and in pathology coincide with hypoxia. The effects of HIF activation may be a facet of normal growth, as in embryonic development, they may counterbalance a disease, as seen in the stimulation of erythropoiesis in anaemia, and they may be part of the pathological processes, as exemplified by tumour angiogenesis. The oxygen-sensitive alpha-subunits of HIF are primarily regulated by the enzymatic hydroxylation that induces rapid proteasomal degradation. The HIFalpha hydroxylases belong to a superfamily of dioxygenases that require the co-substrates oxygen and 2-oxoglutarate as well as the cofactors Fe2+ and ascorbate. The regulation of enzyme turnover by the concentration of the cosubstrate oxygen constitutes the interface between tissue oxygen level and the activity of HIF. The HIFalpha prolyl hydroxylases, termed PHDs/EGLNs (prolyl hydroxylase domain proteins/EGL nine homologues), bind to a conserved Leu-Xaa-Xaa-Leu-Ala-Pro motif present in all substrates identified so far. This recognition motif is present twice in HIF1alpha, which gives rise to a NODD [N-terminal ODD (oxygen-dependent degradation domain)] containing Pro402 of HIF1alpha and a CODD (C-terminal ODD) where Pro564 is hydroxylated. PHD1/EGLN2 and PHD2/EGLN1 hydroxylate both ODDs with higher activity towards CODD, whereas PHD3/EGLN3 is specific for CODD. The reason for this behaviour has been unclear. In this issue of the Biochemical Journal, Villar and colleagues demonstrate that distinct PHD/EGLN domains, that are remote from the catalytic site, function in substrate discrimination. This elegant study improves our understanding of the interaction of the oxygen-sensing PHDs/EGLNs with their substrates, which include, but are not limited to, the HIFalpha proteins.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Oxígeno/química , Oxígeno/metabolismo , Especificidad por Sustrato/fisiologíaRESUMEN
Hypoxia inducible factor-1 (HIF-1) is the master regulator of metabolic adaptation to hypoxia. It is appreciated that HIF-1alpha accumulation is achieved under normoxic conditions by e.g., nitric oxide. We determined molecular mechanisms of HIF-1alpha accumulation under the impact of S-nitrosoglutathione (GSNO). In human embryonic kidney cells GSNO provoked nuclear accumulation of HIF-1alpha. This appeared unrelated to gene transcription and protein translation, thus pointing to inhibition of HIF-1alpha degradation. Indeed, GSNO as well as the hypoxia mimic CoCl2 decreased ubiquitination of HIF-1alpha and GSNO-induced HIF-1alpha failed to coimmunoprecipitate with pVHL (von Hippel Lindau protein). Considering that HIF-1alpha-pVHL interactions require prolyl hydroxylation of HIF-1alpha, we went on to demonstrate inhibition of HIF-1alpha prolyl hydroxylases (PHDs) by GSNO. In vitro HIF-1alpha-pVHL interactions revealed that GSNO dose-dependently inhibits PHD activity but not the interaction of a synthetic peptide resembling the hydroxylated oxygen-dependent degradation domain of HIF-1alpha with pVHL. We conclude that GSNO-attenuated prolyl hydroxylase activity accounts for HIF-1alpha accumulation under conditions of NO formation during normoxia and that PHD activity is subject to regulation by NO.
Asunto(s)
Hipoxia de la Célula , Óxido Nítrico/fisiología , Procolágeno-Prolina Dioxigenasa/metabolismo , S-Nitrosoglutatión/metabolismo , Factores de Transcripción/metabolismo , Células Cultivadas , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Unión Proteica , Biosíntesis de Proteínas , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
Endoplasmic reticulum (ER) stress leads to activation of the unfolded protein response (UPR) that results in transient suppression of protein translation to allow recovery but leads to cell death when stress cannot be resolved. Central to initiation of the UPR is the activation of the ER transmembrane kinase protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). Here we report that the thiol oxidoreductase ERp57 and protein disulfide isomerase-A1 (PDI), which belong to the same family of luminal ER oxidoreductases, have strikingly opposing roles in the regulation of PERK function. In HCT116 colon carcinoma cells, lentiviral depletion of ERp57 resulted in oxidation of PDI and activation of PERK, whereas depletion or chemical inhibition of PDI reduced PERK signaling and sensitized the cancer cells to hypoxia and ER stress. We conclude that oxidized PDI acts as a PERK activator, whereas ERp57 keeps PDI in a reduced state in the absence of ER stress. Thus, our study defines a new interface between metabolic redox signaling and PERK-dependent activation of the UPR and has the potential to influence future cancer therapies that target PERK signaling.