Estabilización de Hif-1α en la cresta neural de pez cebra
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2019
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es
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Universidad Andrés Bello
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El factor inducido por hipoxia-1 (HIF-1) es considerado el regulador maestro de la respuesta celular a hipoxia (baja tensión de oxígeno). HIF-1 presenta un tipo de respuesta rápida en la cual, bajo tensiones normales de oxígeno (normoxia) el factor es degradado vía proteosoma, mientras que en condiciones de hipoxia el factor se estabiliza, permitiendo la activación de genes necesarios para el transporte de oxígeno, cambios en el metabolismo y angiogénesis. El oxígeno es capaz de difundir a través de las células, sin embargo, se ha descrito en modelos de cáncer que en tumores de tamaños mayores a 1 mmel oxígeno es incapaz de difundir eficientemente hacia el interior de éste, provocando un centro hipóxico capaz de estabilizar HIF-1. No obstante, existe una creciente evidencia que indica que HIF-1 también podría estar siendo estabilizado durante normoxia por mecanismos independientes de oxígeno. Ambos mecanismos de estabilización de HIF-1, tanto por hipoxia como en normoxia, han sido estudiados extensamente en líneas celulares mientras que in vivo actualmente existe muy poca evidencia sobre la naturaleza de la estabilización de HIF-1.En modelos de cáncer se ha descrito que la estabilización de HIF-1 lleva a un estado celular llamado efecto Warburg, el cual se caracteriza por un cambio de metabolismo energético de oxidativo a glicolítico fermentativo aun cuando los niveles de oxígenos son normales, estado conocido como glicólisis aeróbica. Estudios in vivo en ratón han mostrado que animales knockout para los genes Hif-1αmueren durante el desarrollo embrionario entre los días 8-11 de gestación (E8-E11), exhibiendo dramáticos defectos cardiovasculares, en la angiogénesis y en el cierre del tubo neural. En nuestro laboratorio se ha reportado otra función de Hif-1α en el desarrollo embrionario, mostrando su importante rol en la migración de las células de la cresta neural (NCCs). En el desarrollo de las células de la cresta neural podemos reconocer dos procesos: 1) la inducción de estas célulasy 2) la migración delas mismas a través del embrión. En nuestro laboratorio se ha descrito que Hif-1α(de Xenopus laevisy Danio rerio) regulados aspectos de la migración de lasNCCs:i)EMT: transición epitelial-mesemquimática, es decir la transformación de las NCCs en un mesénquima móvil desde de un epitelio y ii)Quimotaxis, o migración direccionada de las NCCs a lo largo del embrión. Entonces, el ¿cómo ocurre la estabilización de Hif-1αin vivo en las células de la cresta neural?, aún no ha sido determinado usando un modelo animal. Las NCCs tanto en pez cebra como en Xenopus laevis, se localizan cerca de la superficie del embrión, bajo una única capa de células de la epidermis, esta sola observación sugiere que el oxígeno sería capaz de difundir hasta este territorio y que los mecanismos de estabilización de Hif-1α debieran ser independientes de este gas. Con estos antecedentes nos preguntamos:¿cuáles son los mecanismos que regulan la estabilización Hif-1 durante la migración de la cresta neural? Hipotetizamos que la estabilización de Hif-1 en las células de la cresta neural de pez cebra se debe a dos mecanismos independientes de oxígeno: 1)señales extracelulares, como factores de crecimiento que permitirían la EMT, y 2) el contexto metabólico energético intracelular en estas células, similar a lo descrito para el efecto Warburg, que mantendría a Hif-1estabilizado durante la migración de las NCCs. En esta tesis logramos demostrar que la cresta neural de pez cebra es un tejido expuesto a concentraciones normales de oxígeno. Además, que la falta de función del factor de crecimiento derivado de plaquetas-1aa(Pdgf1aa),afecta tanto la migración de la cresta neural como la activación de genes blancos de Hif-1durante el desarrollo de este tejido, lo que siguiere que la vía Pdgf, en pez cebra, participa de la estabilización de Hif-1independiente de oxígeno. Por otra parte, nuestros datos indican que las NCCs migratoria presentan expresión aumentada de transcritos de genes glicolíticos, comparado con la expresión de transcritos de genes del ciclo de ácidos tricarboxílicos (TCA), y que poblaciones celulares dentro de mismo tejido presentan una mayor captación de glucosa, sugiriendo que las NCCs podrían presentar metabolismo de glicólisis aeróbica. Con estos datos proponemos que el cambio metabólico hacia la glicólisis aeróbica(estado similar al efecto Warburg), promueve la disminución de metabolitos del TCA, que resultaría en la estabilización de Hif-1y en la correcta migración de las NCCs. En este contexto, observamos que embriones expuestos al aumento de2-oxoglutarato, un intermediario del TCA, mostraron defectos en la migración de la NCCs; fenotipos que fueron rescatados con un mutante de Hif-1insensible a degradación. Estos datos apoyan que es necesaria la reducción de2-oxoglutarato(TCA), para la correcta migración de las NCCs,sugiriendo una función del metabolismo energético durante el desarrollo de este tejido.
The hypoxia inducible factor (HIF-1) isconsideredthe master regulator of the cellular response to hypoxia (low oxygen tension). HIF-1 presents a type of rapid response in which, under normal oxygen tensions (normoxia) the factor is degraded via proteasome, while under hypoxia the factor stabilizes promoting the expression of genes necessary for oxygen transport, metabolic changes and angiogenesis.Oxygen is able to diffuse through cells, however, it has been described in cancer models thatintumorsover 1mm size, oxygen is unable to efficiently diffuse into the tumor, causing a hypoxic center able to stabilize HIF-1. However, there is a growing evidence indicating that HIF-1 may also be stabilized during normoxia by oxygen-independent mechanisms. Both mechanisms of Hif-1stabilization, by hypoxia and normoxia, have been extensively studied in cell culture while in vivothere is currently very little evidence on the nature of stabilization of HIF-1.In cancer models it has been described that HIF-1 stabilization leads to a cellular state called Warburg effect, which is characterized by a change in energy metabolism from oxidative to glycolytic and fermentative even when oxygen levels are normal, also known as aerobic glycolysis. in vivostudies in mice have shown that knockout animals for Hif-1αgenes die duringembryonic developmentbetween 8-11 (E8-E11)days, exhibiting dramatic cardiovascular defects,angiogenesis andneural tubeclosure. In our laboratory, another function of Hif-1α has been describedin development, showing its important role in neural crest (NC)migration. Within NCdevelopment we can recognize two processes:1)induction of NCCsand2)migration of thesecells along the embryo. In our laboratory it has been described that Hif-1 regulatestwo aspects of NC migration: i) EMT: epithelial-to-mesemquimal transition, the transformation of NC into mobile mesemquima from an epithelium and ii) Chemotaxis, or the directed migration of NCCs trough the embryo. Then: How does the stabilizationHif-1αin vivo in neural crest cells?Has not yet been determinedin animal models. The zebrafish and Xenopus laevisNCCsare located near the surface of the embryo, under onelayer of epidermal cells, this observation suggests that oxygen is capable of diffusing to this territory and thatmechanisms Hif-1 stabilization could be oxygen-independent.With these data we asked: what are the mechanisms that regulate Hif-1stabilization during the neural crestmigration? We hypothesized that: the stabilization of Hif-1in the neural crest cells is due to two types of oxygen-independent mechanisms: 1) extracellular signal, growth factors,allowing the EMTand 2) intracellular energy metabolism context, as observed in the Warburg effect, which sustain Hif-1stabilization trough NC migration. In this thesis we were able to demonstrate that the neural crest is a tissue with normal oxygen concentrations. Also downregulationof the platelet-derived growth factor-1aa (Pdgf1aa) affects NC migration and the expression of Hif-1transcriptional targets during development.This suggest that Pdgfpathway, in zebrafish,has a role in the oxygen-independent Hif-1stabilization. On the other hand, our data indicates that migratoryNCCshave augmented expression of some glycolytic genes compared with TCA cycle related genes, and some cell populations within this tissuehave a high glucose uptake,suggestingthat NC could have an aerobic glycolysis metabolism. With this data we proposethat a metabolic change to an aerobic glycolysis(Warburg effect-like)state promotes a decrease in TCA metabolites, resulting in Hif-1stabilizationandproper NCCs migration.In this context we observed that 2-oxoglutarate, a primary TCA intermediate,exposedembryos have impaired NC migration, and these phenotypes can be rescued by a Hif-1form degradation-insensitive. These data support the idea that 2-oxoglutarate reduction is necessary NC migration, and allow us to speculate that there is role of energy metabolism in this tissue.
The hypoxia inducible factor (HIF-1) isconsideredthe master regulator of the cellular response to hypoxia (low oxygen tension). HIF-1 presents a type of rapid response in which, under normal oxygen tensions (normoxia) the factor is degraded via proteasome, while under hypoxia the factor stabilizes promoting the expression of genes necessary for oxygen transport, metabolic changes and angiogenesis.Oxygen is able to diffuse through cells, however, it has been described in cancer models thatintumorsover 1mm size, oxygen is unable to efficiently diffuse into the tumor, causing a hypoxic center able to stabilize HIF-1. However, there is a growing evidence indicating that HIF-1 may also be stabilized during normoxia by oxygen-independent mechanisms. Both mechanisms of Hif-1stabilization, by hypoxia and normoxia, have been extensively studied in cell culture while in vivothere is currently very little evidence on the nature of stabilization of HIF-1.In cancer models it has been described that HIF-1 stabilization leads to a cellular state called Warburg effect, which is characterized by a change in energy metabolism from oxidative to glycolytic and fermentative even when oxygen levels are normal, also known as aerobic glycolysis. in vivostudies in mice have shown that knockout animals for Hif-1αgenes die duringembryonic developmentbetween 8-11 (E8-E11)days, exhibiting dramatic cardiovascular defects,angiogenesis andneural tubeclosure. In our laboratory, another function of Hif-1α has been describedin development, showing its important role in neural crest (NC)migration. Within NCdevelopment we can recognize two processes:1)induction of NCCsand2)migration of thesecells along the embryo. In our laboratory it has been described that Hif-1 regulatestwo aspects of NC migration: i) EMT: epithelial-to-mesemquimal transition, the transformation of NC into mobile mesemquima from an epithelium and ii) Chemotaxis, or the directed migration of NCCs trough the embryo. Then: How does the stabilizationHif-1αin vivo in neural crest cells?Has not yet been determinedin animal models. The zebrafish and Xenopus laevisNCCsare located near the surface of the embryo, under onelayer of epidermal cells, this observation suggests that oxygen is capable of diffusing to this territory and thatmechanisms Hif-1 stabilization could be oxygen-independent.With these data we asked: what are the mechanisms that regulate Hif-1stabilization during the neural crestmigration? We hypothesized that: the stabilization of Hif-1in the neural crest cells is due to two types of oxygen-independent mechanisms: 1) extracellular signal, growth factors,allowing the EMTand 2) intracellular energy metabolism context, as observed in the Warburg effect, which sustain Hif-1stabilization trough NC migration. In this thesis we were able to demonstrate that the neural crest is a tissue with normal oxygen concentrations. Also downregulationof the platelet-derived growth factor-1aa (Pdgf1aa) affects NC migration and the expression of Hif-1transcriptional targets during development.This suggest that Pdgfpathway, in zebrafish,has a role in the oxygen-independent Hif-1stabilization. On the other hand, our data indicates that migratoryNCCshave augmented expression of some glycolytic genes compared with TCA cycle related genes, and some cell populations within this tissuehave a high glucose uptake,suggestingthat NC could have an aerobic glycolysis metabolism. With this data we proposethat a metabolic change to an aerobic glycolysis(Warburg effect-like)state promotes a decrease in TCA metabolites, resulting in Hif-1stabilizationandproper NCCs migration.In this context we observed that 2-oxoglutarate, a primary TCA intermediate,exposedembryos have impaired NC migration, and these phenotypes can be rescued by a Hif-1form degradation-insensitive. These data support the idea that 2-oxoglutarate reduction is necessary NC migration, and allow us to speculate that there is role of energy metabolism in this tissue.
Notas
Tesis (Doctor en Biociencias Moleculares)
Palabras clave
Pez Cebra, Factores de Transcripción