RESUMO
Impaired burn wound healing in the elderly represents a major clinical problem. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator that orchestrates the cellular response to hypoxia. Its actions in dermal wounds promote angiogenesis and improve healing. In a murine burn wound model, aged mice had impaired wound healing associated with reduced levels of HIF-1. When gene therapy with HIF-1 alone did not correct these deficits, we explored the potential benefit of HIF-1 gene therapy combined with the intravenous infusion of bone marrow-derived angiogenic cells (BMDACs) cultured with dimethyloxalylglycine (DMOG). DMOG is known to reduce oxidative degradation of HIF-1. The mice treated with a plasmid DNA construct expressing a stabilized mutant form of HIF-1α (CA5-HIF-1α)+BMDACs had more rapid wound closure. By day 17, there were more mice with completely closed wounds in the treated group (χ(2), P=0.05). The dermal blood flow measured by laser Doppler showed significantly increased wound perfusion on day 11. Homing of BMDACs to the burn wound was dramatically enhanced by CA5-HIF-1α gene therapy. HIF-1α mRNA expression in the burn wound was increased after transfection with CA5-HIF-1α plasmid. Our findings offer insight into the pathophysiology of burns in the elderly and point to potential targets for developing new therapeutic strategies.
Assuntos
Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Queimaduras/fisiopatologia , Terapia Genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Transfecção , Adenoviridae/genética , Envelhecimento , Animais , Queimaduras/genética , Queimaduras/terapia , Células Cultivadas , Terapia Combinada , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , CicatrizaçãoRESUMO
Interactions between cancer cells and stromal cells, including blood vessel endothelial cells (BECs), lymphatic vessel endothelial cells (LECs), bone marrow-derived angiogenic cells (BMDACs) and other bone marrow-derived cells (BMDCs) play important roles in cancer progression. Intratumoral hypoxia, which affects both cancer and stromal cells, is associated with a significantly increased risk of metastasis and mortality in many human cancers. Recent studies have begun to delineate the molecular mechanisms underlying the effect of intratumoral hypoxia on cancer progression. Reduced O2 availability induces the activity of hypoxia-inducible factors (HIFs), which activate the transcription of target genes encoding proteins that play important roles in many critical aspects of cancer biology. Included among these are secreted factors, including angiopoietin 2, angiopoietin-like 4, placental growth factor, platelet-derived growth factor B, stem cell factor (kit ligand), stromal-derived factor 1, and vascular endothelial growth factor. These factors are produced by hypoxic cancer cells and directly mediate functional interactions with BECs, LECs, BMDACs and other BMDCs that promote angiogenesis, lymphangiogenesis, and metastasis. In addition, lysyl oxidase (LOX) and LOX-like proteins, which are secreted by hypoxic breast cancer cells, remodel extracellular matrix in the lungs, which leads to BMDC recruitment and metastatic niche formation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Neoplasias da Mama/patologia , Fator 1 Induzível por Hipóxia/fisiologia , Linfangiogênese , Neovascularização Patológica/etiologia , Células Estromais/fisiologia , Animais , Células da Medula Óssea/fisiologia , Neoplasias da Mama/irrigação sanguínea , Comunicação Celular , Movimento Celular , Proliferação de Células , Células Endoteliais/fisiologia , Humanos , Lipoxigenase/fisiologia , Metástase Linfática , Metástase NeoplásicaRESUMO
Most cases of breast cancer (BrCa) mortality are due to vascular metastasis. BrCa cells must intravasate through endothelial cells (ECs) to enter a blood vessel in the primary tumor and then adhere to ECs and extravasate at the metastatic site. In this study we demonstrate that inhibition of hypoxia-inducible factor (HIF) activity in BrCa cells by RNA interference or digoxin treatment inhibits primary tumor growth and also inhibits the metastasis of BrCa cells to the lungs by blocking the expression of angiopoietin-like 4 (ANGPTL4) and L1 cell adhesion molecule (L1CAM). ANGPTL4 is a secreted factor that inhibits EC-EC interaction, whereas L1CAM increases the adherence of BrCa cells to ECs. Interference with HIF, ANGPTL4 or L1CAM expression inhibits vascular metastasis of BrCa cells to the lungs.
Assuntos
Angiopoietinas/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/secundário , Espermidina/análogos & derivados , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Animais , Neoplasias da Mama/irrigação sanguínea , Hipóxia Celular , Linhagem Celular Tumoral , Feminino , Humanos , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Camundongos , Transplante de Neoplasias , Interferência de RNA , Espermidina/metabolismoRESUMO
The maintenance of oxygen homeostasis is critical for survival, and the master regulator of this process in metazoan species is hypoxia-inducible factor 1 (HIF-1), which controls both O(2) delivery and utilization. Under conditions of reduced O(2) availability, HIF-1 activates the transcription of genes, whose protein products mediate a switch from oxidative to glycolytic metabolism. HIF-1 is activated in cancer cells as a result of intratumoral hypoxia and/or genetic alterations. In cancer cells, metabolism is reprogrammed to favor glycolysis even under aerobic conditions. Pyruvate kinase M2 (PKM2) has been implicated in cancer growth and metabolism, although the mechanism by which it exerts these effects is unclear. Recent studies indicate that PKM2 interacts with HIF-1α physically and functionally to stimulate the binding of HIF-1 at target genes, the recruitment of coactivators, histone acetylation, and gene transcription. Interaction with HIF-1α is facilitated by hydroxylation of PKM2 at proline-403 and -408 by PHD3. Knockdown of PHD3 decreases glucose transporter 1, lactate dehydrogenase A, and pyruvate dehydrogenase kinase 1 expression; decreases glucose uptake and lactate production; and increases O(2) consumption. The effect of PKM2/PHD3 is not limited to genes encoding metabolic enzymes because VEGF is similarly regulated. These results provide a mechanism by which PKM2 promotes metabolic reprogramming and suggest that it plays a broader role in cancer progression than has previously been appreciated.
Assuntos
Células/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Animais , Homeostase , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Oxigênio/metabolismoAssuntos
Fator 1 Induzível por Hipóxia/metabolismo , Pulmão , Hipersensibilidade Respiratória/fisiopatologia , Fatores de Transcrição/metabolismo , Regulação para Cima , Alérgenos/imunologia , Animais , Asma/imunologia , Asma/metabolismo , Asma/fisiopatologia , Humanos , Fator 1 Induzível por Hipóxia/genética , Pulmão/imunologia , Pulmão/fisiopatologia , Camundongos , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Fatores de Transcrição/genéticaRESUMO
Adaptation of cancer cells to their microenvironment is an important driving force in the clonal selection that leads to invasive and metastatic disease. O2 concentrations are markedly reduced in many human cancers compared with normal tissue, and a major mechanism mediating adaptive responses to reduced O2 availability (hypoxia) is the regulation of transcription by hypoxia-inducible factor 1 (HIF-1). This review summarizes the current state of knowledge regarding the molecular mechanisms by which HIF-1 contributes to cancer progression, focusing on (1) clinical data associating increased HIF-1 levels with patient mortality; (2) preclinical data linking HIF-1 activity with tumor growth; (3) molecular data linking specific HIF-1 target gene products to critical aspects of cancer biology and (4) pharmacological data showing anticancer effects of HIF-1 inhibitors in mouse models of human cancer.
Assuntos
Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Animais , Humanos , Fator 1 Induzível por Hipóxia/genética , Camundongos , Neoplasias/genéticaRESUMO
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric protein composed of HIF-1alpha and HIF-1alpha subunits, which is activated in response to reduced O2 availability. HIF-1 transactivates genes encoding proteins that are involved in key aspects of the cancer phenotype, including cell immortalization and de-differentiation, stem cell maintenance, genetic instability, glucose uptake and metabolism, pH regulation, autocrine growth/survival, angiogenesis, invasion/metastasis, and resistance to chemotherapy. Increased HIF-1alpha levels, as determined by immunohistochemical analysis of tumor biopsy specimens, is associated with increased mortality in many human cancers. Drugs that inhibit HIF-1 activity and have anti-cancer effects in vivo have been identified and clinical trials are warranted to establish the contexts in which addition of such agents to therapy protocols will result in increased patient survival.
Assuntos
Antineoplásicos/farmacologia , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/genética , Neoplasias/mortalidadeRESUMO
SAG (sensitive to apoptosis gene) or ROC2/RBX2 is the second family member of ROC1/RBX1, a component of SCF (Skp1, Cullin, F-box protein) and VCB (von Hippel-Lindau (VHL), Cullin and Elongin B/C) E3 ubiquitin ligases. SAG protected cells from hypoxia-induced apoptosis when overexpressed. We report here that SAG was subjected to hypoxia induction at the levels of mRNA and protein. Hypoxia induction of SAG was largely HIF-1alpha dependent. A consensus HIF-1-binding site, GCGTG was identified in the first intron of the SAG gene. In response to hypoxia, HIF-1 bound to this site and transactivated SAG expression. SAG transactivation required both the intact binding site in cis and HIF-1alpha in trans. On the other hand, like its family member, ROC1, SAG promoted VHL-mediated HIF-1alpha ubiquitination and degradation, which was significantly inhibited upon small interfering RNA silencing of SAG or ROC1. Furthermore, the endogenous HIF-1alpha at both basal and hypoxia-induced levels was significantly increased upon SAG silencing. Finally, SAG forms in vivo complex with Cul-5 and VHL under hypoxia condition. These results suggest an HIF-1-SAG feedback loop in response to hypoxia, as follows: hypoxia induces HIF-1 to transactivate SAG. Induced SAG then promotes HIF-1alpha ubiquitination and degradation. This feedback loop may serve as a cellular defensive mechanism to reduce potential cytotoxic effects of prolonged HIF-1 activation under hypoxia.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Marcação de Genes , Células HeLa , Humanos , Hipóxia/enzimologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/fisiologiaAssuntos
Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Pneumopatias/metabolismo , Neoplasias/metabolismoRESUMO
This review is divided into three parts: (a) The primary site of oxygen sensing is the carotid body which instantaneously respond to hypoxia without involving new protein synthesis, and is historically known as the first oxygen sensor and is therefore placed in the first section (Lahiri, Roy, Baby and Hoshi). The carotid body senses oxygen in acute hypoxia, and produces appropriate responses such as increases in breathing, replenishing oxygen from air. How this oxygen is sensed at a relatively high level (arterial PO2 approximately 50 Torr) which would not be perceptible by other cells in the body, is a mystery. This response is seen in afferent nerves which are connected synaptically to type I or glomus cells of the carotid body. The major effect of oxygen sensing is the increase in cytosolic calcium, ultimately by influx from extracellular calcium whose concentration is 2 x 10(4) times greater. There are several contesting hypotheses for this response: one, the mitochondrial hypothesis which states that the electron transport from the substrate to oxygen through the respiratory chain is retarded as the oxygen pressure falls, and the mitochondrial membrane is depolarized leading to the calcium release from the complex of mitochondria-endoplasmic reticulum. This is followed by influx of calcium. Also, the inhibitors of the respiratory chain result in mitochondrial depolarization and calcium release. The other hypothesis (membrane model) states that K(+) channels are suppressed by hypoxia which depolarizes the membrane leading to calcium influx and cytosolic calcium increase. Evidence supports both the hypotheses. Hypoxia also inhibits prolyl hydroxylases which are present in all the cells. This inhibition results in membrane K(+) current suppression which is followed by cell depolarization. The theme of this section covers first what and where the oxygen sensors are; second, what are the effectors; third, what couples oxygen sensors and the effectors. (b) All oxygen consuming cells have a built-in mechanism, the transcription factor HIF-1, the discovery of which has led to the delineation of oxygen-regulated gene expression. This response to chronic hypoxia needs new protein synthesis, and the proteins of these genes mediate the adaptive physiological responses. HIF-1alpha, which is a part of HIF-1, has come to be known as master regulator for oxygen homeostasis, and is precisely regulated by the cellular oxygen concentration. Thus, the HIF-1 encompasses the chronic responses (gene expression in all cells of the body). The molecular biology of oxygen sensing is reviewed in this section (Semenza). (c) Once oxygen is sensed and Ca(2+) is released, the neurotransmittesr will be elaborated from the glomus cells of the carotid body. Currently it is believed that hypoxia facilitates release of one or more excitatory transmitters from glomus cells, which by depolarizing the nearby afferent terminals, leads to increases in the sensory discharge. The transmitters expressed in the carotid body can be classified into two major categories: conventional and unconventional. The conventional neurotransmitters include those stored in synaptic vesicles and mediate their action via activation of specific membrane bound receptors often coupled to G-proteins. Unconventional neurotransmitters are those that are not stored in synaptic vesicles, but spontaneously generated by enzymatic reactions and exert their biological responses either by interacting with cytosolic enzymes or by direct modifications of proteins. The gas molecules such as NO and CO belong to this latter category of neurotransmitters and have unique functions. Co-localization and co-release of neurotransmitters have also been described. Often interactions between excitatory and inhibitory messenger molecules also occur. Carotid body contains all kinds of transmitters, and an interplay between them must occur. But very little has come to be known as yet. Glimpses of these interactions are evident in the discussion in the last section (Prabhakar).
Assuntos
Oxigênio/metabolismo , Animais , Monóxido de Carbono/metabolismo , Corpo Carotídeo/metabolismo , Membrana Celular/metabolismo , Humanos , Hipóxia/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Neurotransmissores/metabolismo , Óxido Nítrico/metabolismo , Canais de Potássio/metabolismoRESUMO
The hypoxia-inducible factor 1 (HIF-1) plays a critical role in cellular responses to hypoxia. The aim of the present study was to evaluate which genes are induced by hypoxia, and whether this induction is mediated by HIF-1, by expression microarray analysis of wt and HIF-1alpha null mouse fibroblasts. Forty-five genes were up-regulated by hypoxia and 40 (89%) of these were regulated by HIF-1. Of the 114 genes down-regulated by hypoxia, 19 (17%) were HIF-1-dependent. All glycolytic enzymes were strongly up-regulated by hypoxia in a HIF-1-dependent manner. Genes already known to be related to hypoxia, such as glucose transporter 1, BNIP3, and hypoxia-induced gene 1, were induced. In addition, multiple new HIF-1-regulated genes were identified, including genes involved in metabolism (adenylate kinase 4, galactokinase), apoptosis (galectin-3 and gelsolin), and invasion (RhoA). Genes down-regulated by hypoxia were involved in cytoskeleton maintenance (Rho kinase), mRNA processing (heterogeneous nuclear ribonucleoprotein H1 and splicing factor), and DNA repair (REV3). Furthermore, seven cDNAs from genes with unknown function or expressed sequence tags (ESTs) were up-regulated and 27 such cDNAs were down-regulated. In conclusion, hypoxia causes down- rather than up-regulation of gene expression and HIF-1 seems to play a major role in the regulation of hypoxia-induced genes.
Assuntos
Proteínas de Ligação a DNA/genética , Fibroblastos/fisiologia , Hipóxia/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Regulação para Cima/genética , Animais , Apoptose/genética , Movimento Celular/genética , Citoesqueleto/genética , Reparo do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Regulação para Baixo/genética , Galactoquinase/genética , Galectina 3/genética , Gelsolina/genética , Perfilação da Expressão Gênica/métodos , Transportador de Glucose Tipo 1 , Glicólise/genética , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Proteínas de Membrana/genética , Camundongos , Proteínas de Transporte de Monossacarídeos/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Supramolecular complexes, such as chaperonins, are suitable samples for atomic force microscope structural studies because they have a very well defined shape. High-resolution images can be made using tapping mode in liquid under native conditions. Details about the two-dimensional structures formed onto the surface upon adsorption and of the single protein can be observed. Dissection of the upper ring of the supramolecular complex as a result of the applied lateral force through scanning tip is observed. Finally, the combination of lateral convolution and tip penetration into the cavity of chaperonins offers a direct evaluation of the tip convolution effect on images of macromolecular samples.
Assuntos
Chaperonina 60/química , Microscopia de Força Atômica/métodos , Chaperonas Moleculares/química , Calibragem , Cristalização , Processamento de Imagem Assistida por Computador , Microscopia de Força Atômica/instrumentação , Conformação ProteicaRESUMO
Otto Warburg's classic treatise on the reprogramming of tumour metabolism from oxidative to glycolytic metabolism was published in London in 1930. Although the Warburg effect is one of the most universal characteristics of solid tumours, the molecular basis for this phenomenon has only recently been elucidated by studies indicating that increased expression of genes encoding glucose transporters and glycolytic enzymes in tumour cells is mediated by the transcription factors c-MYC and HIF-1. Whereas c-myc is a direct target for oncogenic mutations, expression of hypoxia-inducible factor 1 (HIF-1) is indirectly up-regulated via gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes that result increased HIF-1alpha protein expression and/or increased HIF-1 transcriptional activity in a cell-type-specific manner. As a result of genetic alterations and intratumoral hypoxia, HIF-1alpha is overexpressed in the majority of common human cancers relative to the surrounding normal tissue. In human breast cancer and brain tumours, HIF-1alpha overexpression is strongly correlated with tumour grade and vascularity.
Assuntos
Neoplasias/metabolismo , Fatores de Transcrição , Hipóxia Celular/fisiologia , Proteínas de Ligação a DNA/genética , Genes myc , Glicólise , Humanos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Mutação , Proteínas Nucleares/genéticaRESUMO
Volatile anesthetics modulate a variety of physiological and pathophysiological responses including hypoxic responses. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates cellular and systemic homeostatic responses to reduced O(2) availability in mammals, including erythropoiesis, angiogenesis, and glycolysis. We demonstrate for the first time that the volatile anesthetic halothane blocks HIF-1 activity and downstream target gene expressions induced by hypoxia in the human hepatoma-derived cell line, Hep3B. Halothane reversibly blocks hypoxia-induced HIF-1alpha protein accumulation and transcriptional activity at clinically relevant doses.
Assuntos
Anestésicos Inalatórios/farmacologia , Halotano/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Anaerobiose , Cobalto/farmacologia , Desferroxamina/farmacologia , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Ativação Transcricional , Células Tumorais CultivadasRESUMO
Hypoxia-inducible factor 1 (HIF-1) is a master regulator of oxygen homeostasis that controls angiogenesis, erythropoiesis, and glycolysis via transcriptional activation of target genes under hypoxic conditions. O(2)-dependent binding of the von Hippel-Lindau (VHL) tumor suppressor protein targets the HIF-1alpha subunit for ubiquitination and proteasomal degradation. The activity of the HIF-1alpha transactivation domains is also O(2) regulated by a previously undefined mechanism. Here, we report the identification of factor inhibiting HIF-1 (FIH-1), a protein that binds to HIF-1alpha and inhibits its transactivation function. In addition, we demonstrate that FIH-1 binds to VHL and that VHL also functions as a transcriptional corepressor that inhibits HIF-1alpha transactivation function by recruiting histone deacetylases. Involvement of VHL in association with FIH-1 provides a unifying mechanism for the modulation of HIF-1alpha protein stabilization and transcriptional activation in response to changes in cellular O(2) concentration.
Assuntos
Hipóxia Celular/fisiologia , Ligases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos , Animais , Células Cultivadas , Primers do DNA/química , Proteínas de Ligação a DNA , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica , Glutationa Transferase/metabolismo , Histona Desacetilases/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Técnicas In Vitro , Luciferases/metabolismo , Oxigenases de Função Mista , Dados de Sequência Molecular , Proteínas Nucleares/genética , Oxigênio/metabolismo , Reação em Cadeia da Polimerase , Conformação Proteica , Coelhos , Proteínas Repressoras/genética , Reticulócitos/metabolismo , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Transcrição Gênica , Ativação Transcricional , Técnicas do Sistema de Duplo-Híbrido , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
Hypoxia-inducible factor 1 (HIF-1) is a global regulator of cellular and systemic O(2) homeostasis in animals. A molecular basis for O(2)-regulated expression of the HIF-1 alpha subunit has now been determined, providing a mechanism for changes in gene expression in response to changes in cellular oxygenation.