La exposición simultánea a dexametasona y rosiglitazona es necesaria y suficiente para generar, a partir de células madre mesenquimáticas, adipocitos funcionales
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2012
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es
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Universidad Andrés Bello
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Resumen
El producto de la adipogénesis es un adipocito funcional, célula que adquiere la maquinaria necesaria para el transporte y síntesis de lípidos, pierde la capacidad de proliferar, tiene alta sensibilidad a insulina y secreta adipoquinas. La etapa mejor caracterizada de la adipogénesis es la maduración, dado que se dispone de líneas celulares comprometidas al linaje adipogénico (3T3-L1 y 3T3-F442A, ambas de ratón). En ellas se han identificado los factores inductores y los mecanismos intracelulares que dan cuentan de la generación de un adipocito funcional. Por el contrario, los eventos moleculares asociados al compromiso de una célula al linaje adipogénico son escasamente conocidos. Las células madre mesenquimáticas (MSCs) representan un atractivo modelo para estudiar las primeras etapas de la adipogénesis, ya que originan, entre otras células, adipocitos. Su multipotencialidad, sus propiedades inmunomoduladoras, y la simpleza con que se aíslan y expanden ex vivo hacen de las MSCs una herramienta ideal para realizar terapia celular. Es así que tanto a nivel pre-clínico como clínico se está evaluando su impacto en diversas patologías.
Actualmente, para inducir la diferenciación de las MSCs al linaje adipogénico, las células se exponen a un glucocorticoide (dexametasona), un inhibidor de fosfodiesterasa (isobutil-metil-xantina), un inhibidor de ciclooxigenasa (indometacina) e insulina, combinación que en adelante llamaremos: coctel clásico (CC). Aunque efectivo, dada su complejidad, este estímulo ha dificultado el estudio de los eventos moleculares asociados al proceso de diferenciación adipogénico. Además, a la fecha se desconoce si los adipocitos generados a partir de MSCs expuestas al CC han completado su maduración. En consecuencia, es de interés científico y biotecnológico disponer de un estímulo constituido por menos componentes, cuyo mecanismo de acción sea más fácil de estudiar y que resulte en la generación de adipocitos funcionales. Durante la unidad de investigación desarrollada antes del inicio de esta tesis evaluamos putativos inductores adipogénicos y determinamos que la exposición simultánea a dexametasona y rosiglitazona (D+R) resulta en la generación de adipocitos a partir de MSCs humanas (hMSCs) y de otras especies de mamíferos (ratón, rata, conejo y perro).
En la presente tesis hemos avanzado en la caracterización de este inductor de diferenciación adipogénica. Para ello determinamos el estado funcional de los adipocitos generados a partir de hMSCs expuestas a D+R y nos aproximarnos al conocimiento de los mecanismos moleculares asociados a la diferenciación adipogénica inducida por D+R en hMSCs. En el contexto del primer objetivo determinamos el perfil de expresión de los genes propios de adipocitos maduros (RTPCR tiempo real), esto es: i) genes asociados a compromiso adipogénico (CEBP-a y PPAR-Y), ii) genes asociados al almacenamiento lipídico (aP2 y LPL), iii) genes asociados al metabolismo de triacilgliceroles (ACS y PEPCK) y iv) genes de adipoquinas (ADIPONECTINA y LEPTINA). Los resultados obtenidos muestran que todos los genes estudiados se expresan en los adipocitos generados a partir de hMSCs expuestas a D+R. También evaluamos su potencial de proliferación (inmunocitofluorescencia para BrdU y citometría de flujo) y su sensibilidad a insulina (incorporación [H 3]2-DG), encontrando que no proliferan y que responden a bajas concentraciones de insulina. Por último determinamos la capacidad de los adipocitos de secretar adipoquinas (ELISA), demostrando que aquellos generados usando D+R secretan adiponectina y leptina a niveles significativamente mayores que los generados usando CC. En consecuencia, los adipocitos generados a partir de hMSCs expuestas a D+R presentan un claro fenotipo de adipocitos funcionales.
Para aproximarnos al mecanismo de acción, primero comprobamos que el blanco canónico de dexametasona, es decir el receptor de glucocorticoides (RG) y él de rosiglitazona, o sea el receptor activado por proliferadores peroxisomales isoforma gamma (PPAR-Y) efectivamente participan en el proceso de diferenciación adipogénico inducido por D+R en las hMSCs. Para ello evaluamos la eficiencia de la generación de adipocitos (microscopia óptica y citometría de flujo) en presencia de un inhibidor de RG (RU486) y/o de un inhibidor de PPAR-Y (GW9662). Los resultados obtenidos muestran que RU486 inhibe totalmente la diferenciación adipogénica mientras que GW9662, lo hace en forma parcial. También observamos inducción de la expresión y translocación desde el citoplasma al núcleo de CEBP-Œ y PPAR-Y, en adipocitos diferenciados con D+R. Finalmente al evaluar la dependencia y la temporalidad de las vías activadas por dexametasona y por rosiglitazona mediadas por C/EBPs y por PPAR-Y , respectivamente; observamos que ellas son interdependientes, y que el tiempo de exposición a dexametasona o rosiglitazona determinan la eficiencia con que ocurre la diferenciación adipogénica de hMSCs inducida por D+R.
Considerando todo lo anterior, en la presente tesis se concluye que la activación simultánea de las vías de señalización canónicas de dexametasona y rosiglitazona es necesaria y suficiente para generar, a partir de hMSCs, adipocitos funcionales. Además se muestra que, comparado con CC, D+R presenta ventajas evidentes tanto respecto de su uso como de su comercialización, siendo estas: i) composición más sencilla, ii) promoción de la diferenciación adipogénica de MSCs de diferentes especies de mamíferos, incluso de aquellas refractarias a CC, y iii) generación de adipocitos funcionales, esto es, células que presentan características propias de los adipocitos que se encuentran in vivo.
The product of adipogenesis is a functional adipocyte. This cell acquires tha necessary machinery for lipid synthesis and transport, loses the ability to proliferate, has high sensitivity to insulin and secretes adipokines. Due to the availability of cell lines committed to the adipogenic lineage (3T3-L1 and 3T3-F442A, both from mouse) maturation has been the most characterized stage of adipogenesis. These cell lines have been used to identify the inducing factors and the intracellular mechanisms that account for the generation of a functional adipocyte. By contrast, the molecular events associated with the commitment of a cell to adipogenic lineage are poorly known. Mesenchymal stem cells (MSCs) represent an attractive model for studying the early stages of adipogenesis, since, among other cells, they could generate adipocytes. Their multipotency, immunomodulatory properties, and the simplicity with they are isolated and ex vivo expanded make MSCs an ideal tool for cell therapy. Thus, the therapeutic potential of MSCs in different pathologies has being evaluated both at pre-clinical and clinical level. Currently, to induce differentiation of MSCs to adipogenic lineage, cell are exposed to a glucocorticoid (dexamethasone), to a phosphor diesterase inhibitor (isobutylmethylxanthine), to a cyclooxigenase inhibitor (indomethacin) and insulin, a combination that from now we will call classic cocktail (CC). Although effective, given its complexity, this stimulation has hampered tha study of molecular events associated with the adipogenic differentiation process. In addition, is unknown whether adipocytes generated from MSCs exposed to the CC completed their maturation. Therefore, is of scientific and biotechnological interest to have an adipogenic stimulus consisting of fewer components, whose mechanism of action could be easier to study and results in the generation of functional adipocytes. During the research unit developed before the start of this thesis, we evaluate putative adipogenic inducers and determined that the simultaneous exposure to dexamethasone and rosiglitazone (D+R) results in the generation of adipocytes from human MSCs (hMScs) and other mammal's species (mouse, rat, rabbit and dog). In this thesis we have made progress in characterizing the adipogenic inducer. To do that we determined the functional status of the adipocytes generated from hMSCs exposed to D+R and we have approximated to understand Wich are the molecular mechanisms associated with adipogenic differentiation induced by D+R in hMSCs. In the context of the first goal we determined the expression profile of genes characteristic of mature adipocytes (by real time RT-PCR), namely: i) genes associated with adipogenic commitment (CEBP-a and PPAR-Y), ii) genes associated with lipid storage (aP2 and LPL), iii) genes associated with the metabolism of triacylglycerols (ACS and PEPCK) and iv) genes coding for adipokines (ADIPONECT/N and LEPT/N). Results show that all the genes studied are expressed in adipocytes generated from hMSCs exposed to D+R. We also evaluated the potential of adipocytes for proliferation (by BrdU incorporation detected by immunocytofluorescence and by flow cytometry) and the insulin sensitivity (by incorporation of [1-1 3] 2-DG). We found that the generate adipocytes do not proliferate and respond to low concentrations of insulin. Finally we determine the ability of adipocytes to secrete adipokines (by ELISA). We showed that those adipocytes generated using D+R secrete adiponectin and leptin at levels significantly higher than those secrete by adipocytes generated using CC. Consequently, adipocytes generated from hMSCs exposed to D+R has a clear functional adipocyte phenotype. In an attempt to approach to the mechanism of action of D+R, first we corroborate that the canonical target of dexamethasone, ie the glucocorticoid receptor (GR) and the canonical target of rosiglitazone , ie the peroxisome proliferator-activated receptor isoform gamma (PPARy) actually participate in the adipogenic differentiation process induced by D+R in hMSCs. For that, we assessed the efficiency of generation of adipocytes (by light microscopy and flow cytometry) in the presence of an inhibitor of GR (RU486) and/or a PPAR-Y inhibitor (GW9662). The results show that RU486 completely inhibits adipogenic differentiation whereas GW9662, only inhibits adipogenic differentiation partially. We also observed induction of expression and translocation from the cytoplasm to the nucleus of CEBP-(I and PPAR-Y, in adipocytes differentiated with D+R. Finally, when we assessed the dependence and timing of the pathways activated by dexamethasone and rosiglitazone mediated by C/EBPs and PPAR-Y, respectively, we saw that they are interdependent, and that the time of exposure to dexamethasone or rosiglitazone determines the final efficiency of the adipogenic differentiation of hMSCs induced by D+R. Considering the above, in this thesis we concluded that simultaneous activation of canonical signaling pathways of dexamethasone and rosiglitazone is necessary and sufficient to generate, from hMSCs functional adipocytes. We also shows that, compared with CC, D+R has obvious advantages both in terms of their use and its commercialization, these are: i) simpler composition, ii) promoting adipogenic differentiation of MSCs from different species of mammals, including those refractory to CC, and iii) generation of functional adipocytes, ie cells with the adipocyte characteristic found in vivo.
The product of adipogenesis is a functional adipocyte. This cell acquires tha necessary machinery for lipid synthesis and transport, loses the ability to proliferate, has high sensitivity to insulin and secretes adipokines. Due to the availability of cell lines committed to the adipogenic lineage (3T3-L1 and 3T3-F442A, both from mouse) maturation has been the most characterized stage of adipogenesis. These cell lines have been used to identify the inducing factors and the intracellular mechanisms that account for the generation of a functional adipocyte. By contrast, the molecular events associated with the commitment of a cell to adipogenic lineage are poorly known. Mesenchymal stem cells (MSCs) represent an attractive model for studying the early stages of adipogenesis, since, among other cells, they could generate adipocytes. Their multipotency, immunomodulatory properties, and the simplicity with they are isolated and ex vivo expanded make MSCs an ideal tool for cell therapy. Thus, the therapeutic potential of MSCs in different pathologies has being evaluated both at pre-clinical and clinical level. Currently, to induce differentiation of MSCs to adipogenic lineage, cell are exposed to a glucocorticoid (dexamethasone), to a phosphor diesterase inhibitor (isobutylmethylxanthine), to a cyclooxigenase inhibitor (indomethacin) and insulin, a combination that from now we will call classic cocktail (CC). Although effective, given its complexity, this stimulation has hampered tha study of molecular events associated with the adipogenic differentiation process. In addition, is unknown whether adipocytes generated from MSCs exposed to the CC completed their maturation. Therefore, is of scientific and biotechnological interest to have an adipogenic stimulus consisting of fewer components, whose mechanism of action could be easier to study and results in the generation of functional adipocytes. During the research unit developed before the start of this thesis, we evaluate putative adipogenic inducers and determined that the simultaneous exposure to dexamethasone and rosiglitazone (D+R) results in the generation of adipocytes from human MSCs (hMScs) and other mammal's species (mouse, rat, rabbit and dog). In this thesis we have made progress in characterizing the adipogenic inducer. To do that we determined the functional status of the adipocytes generated from hMSCs exposed to D+R and we have approximated to understand Wich are the molecular mechanisms associated with adipogenic differentiation induced by D+R in hMSCs. In the context of the first goal we determined the expression profile of genes characteristic of mature adipocytes (by real time RT-PCR), namely: i) genes associated with adipogenic commitment (CEBP-a and PPAR-Y), ii) genes associated with lipid storage (aP2 and LPL), iii) genes associated with the metabolism of triacylglycerols (ACS and PEPCK) and iv) genes coding for adipokines (ADIPONECT/N and LEPT/N). Results show that all the genes studied are expressed in adipocytes generated from hMSCs exposed to D+R. We also evaluated the potential of adipocytes for proliferation (by BrdU incorporation detected by immunocytofluorescence and by flow cytometry) and the insulin sensitivity (by incorporation of [1-1 3] 2-DG). We found that the generate adipocytes do not proliferate and respond to low concentrations of insulin. Finally we determine the ability of adipocytes to secrete adipokines (by ELISA). We showed that those adipocytes generated using D+R secrete adiponectin and leptin at levels significantly higher than those secrete by adipocytes generated using CC. Consequently, adipocytes generated from hMSCs exposed to D+R has a clear functional adipocyte phenotype. In an attempt to approach to the mechanism of action of D+R, first we corroborate that the canonical target of dexamethasone, ie the glucocorticoid receptor (GR) and the canonical target of rosiglitazone , ie the peroxisome proliferator-activated receptor isoform gamma (PPARy) actually participate in the adipogenic differentiation process induced by D+R in hMSCs. For that, we assessed the efficiency of generation of adipocytes (by light microscopy and flow cytometry) in the presence of an inhibitor of GR (RU486) and/or a PPAR-Y inhibitor (GW9662). The results show that RU486 completely inhibits adipogenic differentiation whereas GW9662, only inhibits adipogenic differentiation partially. We also observed induction of expression and translocation from the cytoplasm to the nucleus of CEBP-(I and PPAR-Y, in adipocytes differentiated with D+R. Finally, when we assessed the dependence and timing of the pathways activated by dexamethasone and rosiglitazone mediated by C/EBPs and PPAR-Y, respectively, we saw that they are interdependent, and that the time of exposure to dexamethasone or rosiglitazone determines the final efficiency of the adipogenic differentiation of hMSCs induced by D+R. Considering the above, in this thesis we concluded that simultaneous activation of canonical signaling pathways of dexamethasone and rosiglitazone is necessary and sufficient to generate, from hMSCs functional adipocytes. We also shows that, compared with CC, D+R has obvious advantages both in terms of their use and its commercialization, these are: i) simpler composition, ii) promoting adipogenic differentiation of MSCs from different species of mammals, including those refractory to CC, and iii) generation of functional adipocytes, ie cells with the adipocyte characteristic found in vivo.
Notas
Tesis (Doctor en Biotecnología)
Palabras clave
Células Madre, Investigaciones, Dexametasona