RESUMEN
AIM: Constitutive genetic deletion of the adaptor protein p66(Shc) was shown to protect from ischaemia/reperfusion injury. Here, we aimed at understanding the molecular mechanisms underlying this effect in stroke and studied p66(Shc) gene regulation in human ischaemic stroke. METHODS AND RESULTS: Ischaemia/reperfusion brain injury was induced by performing a transient middle cerebral artery occlusion surgery on wild-type mice. After the ischaemic episode and upon reperfusion, small interfering RNA targeting p66(Shc) was injected intravenously. We observed that post-ischaemic p66(Shc) knockdown preserved blood-brain barrier integrity that resulted in improved stroke outcome, as identified by smaller lesion volumes, decreased neurological deficits, and increased survival. Experiments on primary human brain microvascular endothelial cells demonstrated that silencing of the adaptor protein p66(Shc) preserves claudin-5 protein levels during hypoxia/reoxygenation by reducing nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. Further, we found that in peripheral blood monocytes of acute ischaemic stroke patients p66(Shc) gene expression is transiently increased and that this increase correlates with short-term neurological outcome. CONCLUSION: Post-ischaemic silencing of p66(Shc) upon reperfusion improves stroke outcome in mice while the expression of p66(Shc) gene correlates with short-term outcome in patients with ischaemic stroke.
Asunto(s)
Lesiones Encefálicas/prevención & control , Silenciador del Gen/fisiología , Daño por Reperfusión/prevención & control , Proteínas Adaptadoras de la Señalización Shc/genética , Accidente Cerebrovascular/prevención & control , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Animales , Barrera Hematoencefálica/fisiología , Estudios de Casos y Controles , Células Cultivadas , Claudina-5/efectos de los fármacos , Células Endoteliales/fisiología , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Infarto de la Arteria Cerebral Media , Poscondicionamiento Isquémico/métodos , Masculino , Ratones Endogámicos C57BL , Microcirculación/fisiología , Persona de Mediana Edad , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Especies Reactivas de Oxígeno/farmacología , Proteínas Adaptadoras de la Señalización Shc/fisiología , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Resultado del TratamientoRESUMEN
Tumor progression is intrinsically tied to the clonal selection of tumor cells with acquired phenotypes allowing to cope with a hostile microenvironment. Hypoxia-inducible factors (HIFs) master the transcriptional response to local tissue hypoxia, a hallmark of solid tumors. Here, we report significantly longer patient survival in breast cancer with high levels of HIF-2α. Amphiregulin (AREG) and WNT1-inducible signaling pathway protein-2 (WISP2) expression was strongly HIF-2α-dependent and their promoters were particularly responsive to HIF-2α. The endogenous AREG promoter recruited HIF-2α in the absence of a classical HIF-DNA interaction motif, revealing a novel mechanism of gene regulation. Loss of AREG expression in HIF-2α-depleted cells was accompanied by reduced activation of epidermal growth factor (EGF) receptor family members. Apparently opposing results from patient and in vitro data point to an HIF-2α-dependent auto-stimulatory tumor phenotype that, while promoting EGF signaling in cellular models, increased the survival of diagnosed and treated human patients. Our findings suggest a model where HIF-2α-mediated autocrine growth signaling in breast cancer sustains a state of cellular self-sufficiency, thereby masking unfavorable microenvironmental growth conditions, limiting adverse selection and improving therapy efficacy. Importantly, HIF-2α/AREG/WISP2-expressing tumors were associated with luminal tumor differentiation, indicative of a better response to classical treatments. Shifting the HIF-1/2α balance toward an HIF-2-dominated phenotype could thus offer a novel approach in breast cancer therapy.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias de la Mama/metabolismo , Proteínas CCN de Señalización Intercelular/metabolismo , Receptores ErbB/metabolismo , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Represoras/metabolismo , Anfirregulina , Comunicación Autocrina , Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Neoplasias de la Mama/terapia , Hipoxia de la Célula , Línea Celular Tumoral , Proliferación Celular , Supervivencia sin Enfermedad , Familia de Proteínas EGF , Factor de Crecimiento Epidérmico/metabolismo , Femenino , Humanos , Receptor ErbB-4 , Transducción de SeñalRESUMEN
Hypoxia-elicited adaptations of tumor cells are essential for tumor growth and cancer progression. Although ample evidence exists for a positive correlation between hypoxia-inducible factors (HIFs) and tumor formation, metastasis and bad prognosis, the function of the HIF-α protein stability regulating prolyl-4-hydroxylase domain enzyme PHD2 in carcinogenesis is less well understood. In this study, we demonstrate that downregulation of PHD2 leads to increased tumor growth in a hormone-dependent mammary carcinoma mouse model. Tissue microarray analysis of PHD2 protein expression in 281 clinical samples of human breast cancer showed significantly shorter survival times of patients with low-level PHD2 tumors over a period of 10 years. An angiogenesis-related antibody array identified, amongst others, amphiregulin to be increased in the absence of PHD2 and normalized after PHD2 reconstitution. Cultivation of endothelial cells in conditioned media derived from PHD2-downregulated cells resulted in enhanced tube formation that was blocked by the addition of neutralizing anti-amphiregulin antibodies. Functionally, amphiregulin was regulated on the transcriptional level specifically by HIF-2 but not HIF-1. Our data suggest that PHD2/HIF-2/amphiregulin signaling has a critical role in the regulation of breast tumor progression and propose PHD2 as a potential tumor suppressor in breast cancer.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias de la Mama/metabolismo , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Anfirregulina , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Progresión de la Enfermedad , Familia de Proteínas EGF , Femenino , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/genética , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Immunoblotting , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/genética , Estimación de Kaplan-Meier , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos , Ratones Desnudos , Persona de Mediana Edad , Procolágeno-Prolina Dioxigenasa/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante HeterólogoRESUMEN
The onconeuronal cerebellar degeneration-related antigen Cdr2 is associated with paraneoplastic syndromes. Neoplastic expression of Cdr2 in ovary and breast tumors triggers an autoimmune response that suppresses tumor growth by developing tumor immunity, but culminates in cerebellar degeneration when Cdr2-specific immune cells recognize neuronal Cdr2. We identified Cdr2 as a novel interactor of the hypoxia-inducible factor (HIF) prolyl-4-hydroxylase PHD1 and provide evidence that Cdr2 might represent a novel important tumor antigen in renal cancer. Strong Cdr2 protein expression was observed in 54.2% of papillary renal cell carcinoma (pRCC) compared with 7.8% of clear-cell RCC and no staining was observed in chromophobe RCC or oncocytoma. High Cdr2 protein levels correlated with attenuated HIF target gene expression in these solid tumors, and Cdr2 overexpression in tumor cell lines reduced HIF-dependent transcriptional regulation. This effect was because of both attenuation of hypoxic protein accumulation and suppression of the transactivation activity of HIF-1alpha. pRCC is known for its tendency to avascularity, usually associated with a lower pathological stage and higher survival rates. We provide evidence that Cdr2 protein strongly accumulates in pRCC, attenuates the HIF response to tumor hypoxia and may become of diagnostic importance as novel renal tumor marker.
Asunto(s)
Carcinoma de Células Renales/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Hipoxia de la Célula/genética , Línea Celular Tumoral , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Renales/inmunología , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Ratones , Proteínas del Tejido Nervioso/inmunología , Oxígeno , Procolágeno-Prolina Dioxigenasa/metabolismo , Unión Proteica , Transcripción Genética , Activación Transcripcional , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The PAS domain kinase PASKIN, also termed PAS kinase or PASK, is an evolutionarily conserved potential sensor kinase related to the heme-based oxygen sensors of nitrogen-fixing bacteria. In yeast, the two PASKIN homologs link energy flux and protein synthesis following specific stress conditions. In mammals, PASKIN may regulate glycogen synthesis and protein translation. Paskin knock-out mice do not show any phenotype under standard animal husbandry conditions. Interestingly, these mice seem to be protected from the symptoms of the metabolic syndrome when fed a high-fat diet. Energy turnover might be increased in specific PASKIN-deficient cell types under distinct environmental conditions. According to the current model, binding of a putative ligand to the PAS domain disinhibits the kinase domain and activates PASKIN auto- and target phosphorylation. Future research needs to be conducted to elucidate the nature of the putative ligand and the molecular mechanisms of downstream signalling by PASKIN.
Asunto(s)
Metabolismo Energético , Homeostasis , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Grasas de la Dieta , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Insulina/metabolismo , Síndrome Metabólico/metabolismo , Ratones , Modelos Moleculares , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/fisiologíaRESUMEN
Solid tumors often have an inadequate blood supply, which results in large regions that are subjected to hypoxic or anoxic stress. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates much of the transcriptional response of cells to hypoxia. Activating transcription factor 3 (ATF3) is another transcription factor that responds to a variety of stresses and is often upregulated in cancer. We investigated the regulation of ATF3 by oxygen deprivation. ATF3 induction occurred most robustly under anoxia, is common, and it is not dependent on presence of HIF-1 or p53, but is sensitive to the inhibition of c-Jun NH2-terminal kinase activation and the antioxidant N-acetylcystein. ATF3 could also be induced by desferrioxamine but not by the mitochondrial poison cyanide or the nonspecific 2-oxoglutarate dioxygenase inhibitor dimethyloxalylglycine. We also show that anoxic ATF3 mRNA is more stable than normoxic mRNA providing a mechanism for this induction. Thus, this study demonstrates that the regulation of ATF3 under anoxia is independent of 2-oxoglutarate dioxygenase, HIF-1 and p53, presumably involving multiple regulatory pathways.
Asunto(s)
Factor de Transcripción Activador 3/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetilcisteína/farmacología , Factor de Transcripción Activador 3/genética , Aminoácidos Dicarboxílicos/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células Cultivadas/metabolismo , Células Cultivadas/patología , Cianuros/farmacología , Deferoxamina/farmacología , Activación Enzimática , Depuradores de Radicales Libres/farmacología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Melanoma/metabolismo , Melanoma/patología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Oxigenasas de Función Mixta/metabolismo , Neuronas/metabolismo , Neuronas/patología , Oxígeno/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Sideróforos/farmacología , Factores de Transcripción/genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Every cell of the body is capable of measuring the environmental oxygen concentration. When oxygen supply falls below oxygen demand of the cell (hypoxia), a series of genes will be activated whose products help the cell, the tissue as well as the whole organism in adapting to these altered oxygen concentrations. The discovery of hypoxia-inducible transcription factors (HIFs) allowed the molecular understanding of these processes.
Asunto(s)
Hipoxia de la Célula/genética , Regulación de la Expresión Génica , Hipoxia/genética , Consumo de Oxígeno/fisiología , Oxígeno/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Modelos Biológicos , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Conformación Proteica , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
The transforming growth factor-beta 3 (TGF-beta 3) is involved in oxygen-dependent differentiation processes during placental development and pregnancy disorders. However, the importance of oxygen partial pressure for the regulation of TGF-beta 3 expression is presently unclear. We and others presented preliminary evidence that the hypoxia-inducible factor-1 (HIF-1) confers TGF-beta 3 transcription but it was unknown whether this occurred directly or indirectly. To analyze how HIF-1 regulates TGF-beta 3 gene transcription, we cloned and sequenced the mouse TGF-beta 3 promoter region. Multiple putative HIF-1 binding sites (HBSs) were identified, many of which co-localized with two G+C rich CpG islands 5' to the TGF-beta 3 transcription start site. A 6.8 kb fragment of the TGF-beta 3 promoter induced reporter gene expression under hypoxic conditions or when treated with an iron chelator known to stabilize and activate the HIF-1 alpha subunit. Deletion of a 2.4 kb fragment upstream of the distal CpG island abolished inducibility of reporter gene expression. Two HBSs (HBS1 and HBS6) that bound the HIF-1 protein could be identified within this 2.4 kb fragment. These results suggest that TGF-beta 3 gene expression is directly regulated by HIF-1.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/fisiología , Factores de Transcripción , Activación Transcripcional , Factor de Crecimiento Transformador beta/genética , Animales , Sitios de Unión , Diferenciación Celular , Hipoxia de la Célula , ADN/metabolismo , Femenino , Regulación de la Expresión Génica , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Ratones , Embarazo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Factor de Crecimiento Transformador beta3 , Trofoblastos/citologíaRESUMEN
The key elements of circadian clockwork and oxygen homeostasis are the PAS protein family members PER and CLOCK and hypoxia-inducible factor 1alpha (HIF-1alpha). The PAS domain serves as an interface for protein-protein interactions. We asked whether a cross-talk exists between the PAS components of hypoxic and circadian pathways. We found several isoforms of PER1 protein that exhibit tissue-specific size differences. In the mouse brain, a predominantly nuclear 48 kDa isoform that followed a daily rhythm was observed. The 48 kDa form was found in the nuclear fractions derived from mouse liver, Swiss3T3 fibroblasts, and N2A neuroblastoma cells. In mouse kidney and human 293 kidney cells, a 55 kDa PER1 form was detected. CLOCK was observed as a predicted 100 kDa protein in rat-1 cells and in all analyzed mouse tissues including brain, liver, kidney, and spleen. In contrast to PER1, CLOCK protein expression was not rhythmic. Exposure to hypoxia led to increased PER1 and CLOCK protein levels in mice. Based on coimmunoprecipitation experiments that showed protein-protein interaction between PER1 and the alpha subunit of HIF-1, we suggest that these hypoxic effects may be modulated by HIF-1alpha.-Chilov, D., Hofer, T., Bauer, C., Wenger, R. H., Gassmann, M. Hypoxia affects expression of circadian genes PER1 and CLOCK in mouse brain.
Asunto(s)
Encéfalo/metabolismo , Ritmo Circadiano/fisiología , Hipoxia/fisiopatología , Proteínas Nucleares/genética , Transactivadores/genética , Células 3T3 , Animales , Northern Blotting , Western Blotting , Proteínas CLOCK , Proteínas de Ciclo Celular , Línea Celular , Núcleo Celular/metabolismo , Ritmo Circadiano/genética , Dimerización , Regulación de la Expresión Génica , Células HeLa , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Ratones , Ratones Endogámicos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Pruebas de Precipitina , Isoformas de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated alpha subunit and a constitutively expressed beta subunit. Although HIF-1 is regulated mainly by oxygen tension through the oxygen-dependent degradation of its alpha subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF-1 activation in vivo, we determined the spatial and temporal distribution of HIF-1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohistochemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF-1alpha is present in mice kept under normoxic conditions and is further increased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF-1alpha expression varies among different organs. In liver and kidney, HIF-1alpha reaches maximal levels after 1 h and gradually decreases to baseline levels after 4 h of continuous hypoxia. In the brain, however, HIF-1alpha is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF-1beta is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF-1alpha. Overall, HIF-1alpha expression in normoxic mice suggests that HIF-1 has an important role in tissue homeostasis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Hipoxia/fisiopatología , Proteínas Nucleares/metabolismo , Factores de Transcripción , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Núcleo Celular/química , Núcleo Celular/efectos de los fármacos , Citoplasma/química , Citoplasma/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta a Droga , Eritropoyetina/sangre , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Immunoblotting , Inmunohistoquímica , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/efectos de los fármacos , Proteínas Nucleares/genética , Oxígeno/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Distribución TisularRESUMEN
The PAS domain is a versatile protein fold found in many archaeal, bacterial, and plant proteins capable of sensing environmental changes in light intensity, oxygen concentration, and redox potentials. The oxygen sensor FixL from Rhizobium species contains a heme-bearing PAS domain and a histidine kinase domain that couples sensing to signaling. We identified a novel mammalian PAS protein (PASKIN) containing a domain architecture resembling FixL. PASKIN is encoded by an evolutionarily conserved single-copy gene which is ubiquitously expressed. The human PASKIN and mouse Paskin genes show a conserved intron-exon structure and share their promoter regions with another ubiquitously expressed gene that encodes a regulator of protein phosphatase-1. The 144-kDa PASKIN protein contains a PAS region homologous to the FixL PAS domain and a serine/threonine kinase domain which might be involved in signaling. Thus, PASKIN is likely to function as a mammalian PAS sensor protein.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Hemoproteínas/química , Hemoproteínas/metabolismo , Oxígeno/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada/genética , Exones/genética , Dosificación de Gen , Expresión Génica , Histidina Quinasa , Humanos , Intrones/genética , Ratones , Datos de Secuencia Molecular , Peso Molecular , Regiones Promotoras Genéticas/genética , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , SolubilidadRESUMEN
Hypoxia-inducible factors (HIFs) locate to HIF-binding sites (HBSs) within the hypoxia-response elements (HREs) of oxygen-regulated genes. Whereas HIF-1alpha is expressed ubiquitously, HIF-2alpha is found primarily in the endothelium, similar to endothelin-1 (ET-1) and fms-like tyrosine kinase 1 (Flt-1), the expression of which is controlled by HREs. We identified an unique sequence alteration in both ET-1 and Flt-1 HBSs not found in other HIF-1 target genes, implying that these HBSs might cause binding of HIF-2 rather than HIF-1. However, electrophoretic mobility shift assays showed HIF-1 and HIF-2 DNA complex formation with the unique ET-1 HBS to be about equal. Both DNA-binding and hypoxic activation of reporter genes using the ET-1 HBS was decreased compared with transferrin and erythropoietin HBSs. The Flt-1 HBS was non-functional when assayed in isolation, suggesting that additional factors are required for hypoxic up-regulation via the reported Flt-1 HRE. Interestingly, HIF-1 activity could be restored fully by point-mutating the ET-1 (but not the Flt-1) HBS, suggesting that the wild-type ET-1 HBS attenuated the full hypoxic response known from other oxygen-regulated genes. Such a mechanism might serve to limit the expression of this potent vasoconstrictor in hypoxia.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Endotelina-1/metabolismo , Proteínas Nucleares/fisiología , Secuencia de Bases/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Sitios de Unión/genética , Unión Competitiva , Hipoxia de la Célula/genética , Proteínas de Unión al ADN/metabolismo , Endotelina-1/genética , Eritropoyetina/metabolismo , Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Proteínas Nucleares/metabolismo , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Elementos de Respuesta/fisiología , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Receptor 1 de Factores de Crecimiento Endotelial VascularRESUMEN
The heterodimeric hypoxia-inducible factor (HIF)-1 is a master transcriptional regulator of oxygen homeostasis and a possible target for gene therapy of ischemic disease. Although the role of oxygen concentration in HIF-1a protein stabilization is well established, it is less clear whether and how oxygen-regulated mechanisms contribute to HIF-1a protein modifications, nuclear translocation, heterodimerization with the b-subunit, recruitment of cofactors, and gene trans-activation. Because the HIF-1a protein is proteolytically degraded under normoxic conditions, we established two HeLa Tet-Off cell lines (HT42 and HT43), which inducibly overexpress high levels of HIF-1a under normoxic conditions, allowing to distinguish hypoxia-dependent from hypoxia-independent activation mechanisms. Using these cells, we found that normoxically induced HIF-1a is localized to the nucleus, binds DNA, and trans-activates reporter and endogenous target genes. The levels of p53 expression remained unaffected. The MAP kinase inhibitor PD98059 attenuated HIF-1a protein modifications and trans-activation ability but not protein stabilization and DNA-binding activity. Because overexpressed HIF-1a is fully localized to the nucleus but displays only partial DNA-binding and trans-activation activity, mitogen-activated protein kinase-dependent phosphorylation might be required for full HIF-1 activation. HIF-1a protein was also overexpressed in vivo, following the transplantation of HT42 cells into nude mice, demonstrating the feasibility of HIF-1a gene transfer.
Asunto(s)
Hipoxia de la Célula/fisiología , Factores de Transcripción/genética , Animales , Transporte Biológico , Núcleo Celular/metabolismo , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Expresión Génica , Regulación de la Expresión Génica , Terapia Genética/métodos , Transportador de Glucosa de Tipo 1 , Células HeLa , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Linfocinas/genética , Linfocinas/metabolismo , MAP Quinasa Quinasa 1 , Ratones , Ratones Desnudos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Trasplante de Neoplasias , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional/efectos de los fármacos , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularAsunto(s)
Células Quimiorreceptoras/fisiología , Ligasas , Oxígeno/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas/fisiología , Transactivadores/fisiología , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauAsunto(s)
Hipoxia de la Célula/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Oxígeno/metabolismo , Factores de Transcripción/metabolismo , Liasas de Carbono-Oxígeno/metabolismo , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Immunoblotting , Cinética , FN-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Tiorredoxinas/metabolismo , Factores de Tiempo , Factores de Transcripción/genéticaRESUMEN
Nitric oxide (NO) induces vasodilatatory, antiaggregatory, and antiproliferative effects in vitro. To delineate potential beneficial effects of NO in preventing vascular disease in vivo, we generated transgenic mice overexpressing human erythropoietin. These animals induce polyglobulia known to be associated with a high incidence of vascular disease. Despite hematocrit levels of 80%, adult transgenic mice did not develop hypertension or thromboembolism. Endothelial NO synthase levels, NO-mediated endothelium-dependent relaxation and circulating and vascular tissue NO levels were markedly increased. Administration of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) led to vasoconstriction of peripheral resistance vessels, hypertension, and death of transgenic mice, whereas wild-type siblings developed hypertension but did not show increased mortality. L-NAME-treated polyglobulic mice revealed acute left ventricular dilatation and vascular engorgement associated with pulmonary congestion and hemorrhage. In conclusion, we here unequivocally demonstrate that endothelial NO maintains normotension, prevents cardiovascular dysfunction, and critically determines survival in vivo under conditions of increased hematocrit.
Asunto(s)
Enfermedades Cardiovasculares/prevención & control , Eritropoyetina/fisiología , Óxido Nítrico/fisiología , Animales , Inhibidores Enzimáticos/administración & dosificación , Eritropoyetina/genética , Técnicas In Vitro , Ratones , Ratones Transgénicos , NG-Nitroarginina Metil Éster/administración & dosificación , Nitratos/sangre , Óxido Nítrico Sintasa/antagonistas & inhibidores , Análisis de SupervivenciaRESUMEN
The heterodimeric hypoxia-inducible factor-1 (HIF-1), consisting of the subunits HIF-1alpha and HIF-1beta/ARNT, is a master transcriptional regulator of oxygen homeostasis. Under hypoxic conditions, HIF-1alpha levels very rapidly increase, mostly due to protein stabilization. However, translational regulation of HIF-1alpha has not been directly analyzed so far. Mouse HIF-1alpha exists as two mRNA isoforms (termed mHIF-1alphaI.1 and mHIF-1alphaI. 2) containing structurally different 5'-termini which might modulate translation initiation. Whereas the in vitro translation efficiency of these two mRNA isoforms was about equal, the mHIF-1alphaI.2 5'-untranslated region (5'-UTR) conferred significantly higher in vivo luciferase reporter gene activity than the mHIF-1alphaI.1 5'-UTR. Similar corresponding luciferase mRNA levels indicate translational rather than transcriptional alterations. Reporter gene expression was not affected upon exposure of transiently transfected cells to hypoxia (1% oxygen). Direct assessment of translational regulation by polysomal profile analysis of HeLaS3 cells showed that HIF-1alpha (and to a lower extent ARNT) mRNA was found mainly in the translationally active polyribosomal fractions under both normoxic and hypoxic conditions. In contrast, the association of mRNAs for beta-actin and ribosomal protein L28 with the polyribosomal fractions was substantially reduced under hypoxic conditions, suggesting decreased overall protein synthesis. Thus, efficient translation of mouse HIF-1alpha in a situation where the general translation efficiency is reduced represents a prerequisite for the very rapid accumulation of HIF-1alpha protein upon exposure to hypoxia.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas Nucleares/biosíntesis , Receptores de Hidrocarburo de Aril , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo , Hipoxia de la Célula , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Exones , Regulación de la Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Ratones , Proteínas Nucleares/genética , Plásmidos , Biosíntesis de Proteínas , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , ARN Mensajero/biosíntesis , Factores de Transcripción/genética , TransfecciónRESUMEN
Induction of erythropoietin (Epo) expression under hypoxic conditions is mediated by the heterodimeric hypoxia-inducible factor (HIF)-1. Following binding to the 3' hypoxia-response element (HRE) of the Epo gene, HIF-1 markedly enhances Epo transcription. To facilitate the search for HIF-1 (ant)agonists, a hypoxia-reporter cell line (termed HRCHO5) was constructed containing a stably integrated luciferase gene under the control of triplicated heterologous HREs. Among various agents tested, we identified a class of substances called epolones, which induced HRE-dependent reporter gene activity in HRCHO5 cells. Epolones are fungal products known to induce Epo expression in hepatoma cells. We found that epolones (optimal concentration 4-8 micromol/L) potently induce HIF-1 alpha protein accumulation and nuclear translocation as well as HIF-1 DNA binding and reporter gene transactivation. Interestingly, the activity of a compound related to the fungal epolones, ciclopirox olamine (CPX), was blocked after addition of ferrous iron. This suggests that CPX might interfere with the putative heme oxygen sensor, as has been proposed for the iron chelator deferoxamine mesylate (DFX). However, about 10-fold higher concentrations of DFX (50-100 micromol/L) than CPX were required to maximally induce reporter gene activity in HRCHO5 cells. Moreover, structural, functional, and spectrophotometric data imply a chelator:iron stoichiometry of 1:1 for DFX but 3:1 for CPX. Because the iron concentration in the cell culture medium was determined to be 16 micromol/L, DFX but not CPX function can be explained by complete chelation of medium iron. These results suggest that the lipophilic epolones might induce HIF-1 alpha by intracellular iron chelation. (Blood. 2000;96:1558-1565)
Asunto(s)
Proteínas de Unión al ADN/genética , Eritropoyetina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Nucleares/genética , Piridonas/farmacología , Animales , Células CHO , Hipoxia de la Célula , Cricetinae , Proteínas de Unión al ADN/metabolismo , Eritropoyetina/biosíntesis , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Fetal skin wounds heal scarlessly while adult wounds scar. Fetal wound healing occurs in a physiologically hypoxic environment whereas in adult wound healing, cells have to acutely adapt to hypoxia caused by locally impaired blood supply. We examined the expression of hypoxia-inducible factor 1 (HIF-1), a potent transcriptional regulator of oxygen-dependent genes such as vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGF-beta), a potentially HIF-1-regulated scarring cytokine, on fetal and adult responses to wounding. Incisional skin wounds were created in four sheep fetuses (twins served as controls) and two ewes at 100 days of gestation (term = 150 days). Fetal and adult wounds as well as non-wounded control tissues were harvested 2 days post-wounding. Intraoperative arterial blood gas analyses and invasive subcutaneous pO2 measurements revealed that the fetuses were indeed hypoxic while the mothers were normoxic. Expression patterns of HIF-1alpha were investigated by Western blot analyses. HIF-1alpha expression in fetal wounds and fetal control skin was similar, whereas HIF-1alpha was only detected in adult wounds but not in adult control skin. Exposure of cultured fetal and adult dermal fibroblasts to hypoxia (1% O2) showed a marked induction of VEGF mRNA. In contrast, exposure of these cell types to hypoxia did not significantly affect TGF-beta1 mRNA expression in comparison to their normoxic controls. The presence of HIF-1alpha in fetal but not in adult normal skin indicates that HIF-1alpha might be involved in fetal skin development. Conversely, the upregulation of HIF-1alpha in adult but not early fetal wound repair might represent a pathway in the pathogenesis of scarring, since several growth factors overexpressed in, and associated, with scarring are hypoxia-inducible. Further studies need to be performed in order to identify hypoxia-regulated HIF-1alpha target genes involved in the pathogenesis of scarring.