RESUMO
Extracellular factors and intracellular signaling pathways involved in early events of adipocyte differentiation are poorly defined. It is shown herein that expression of leukemia inhibitory factor (LIF) and LIF receptor is developmentally regulated during adipocyte differentiation. Preadipocytes secrete bioactive LIF, and an antagonist of LIF receptor inhibits adipogenesis. Genetically modified embryonic stem (ES) cells combined with culture conditions to commit stem cells into the adipocyte lineage were used to examine the requirement of LIF receptor during in vitro development of adipose cells. The capacity of embryoid bodies derived from lifr(-/-) ES cells to undergo adipocyte differentiation is dramatically reduced. LIF addition stimulates adipocyte differentiation of Ob1771 and 3T3-F442A preadipocytes and that of peroxisome proliferator-activated receptor gamma2 ligand-treated mouse embryonic fibroblasts. Expression of the early adipogenic transcription factors C/EBPbeta and C/EBPdelta is rapidly stimulated following exposure of preadipose cells to LIF. The selective inhibitors of mitogen-activated protein kinase kinase, i.e. PD98059 and U0126, inhibit LIF-induced C/EBP gene expression and prevent adipocyte differentiation induced by LIF. These results are in favor of a model that implicates stimulation of LIF receptor in the commitment of preadipocytes to undergo terminal differentiation by controlling the early expression of C/EBPbeta and C/EBPdelta genes via the mitogen-activated protein kinase cascade.
Assuntos
Adipócitos/metabolismo , Inibidores do Crescimento/metabolismo , Interleucina-6 , Linfocinas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Receptores de Citocinas/metabolismo , Fatores de Transcrição , Adipócitos/citologia , Animais , Proteína delta de Ligação ao Facilitador CCAAT , Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Flavonoides/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Inibidores do Crescimento/genética , Fator Inibidor de Leucemia , Subunidade alfa de Receptor de Fator Inibidor de Leucemia , Linfocinas/genética , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , RNA Mensageiro/metabolismo , Receptores de Citocinas/genética , Receptores de OSM-LIF , Transdução de Sinais , Células-Tronco/metabolismoRESUMO
Embryonic stem cells, derived from the inner cell mass of murine blastocysts, can be maintained in a totipotent state in vitro. In appropriate conditions embryonic stem cells have been shown to differentiate in vitro into various derivatives of all three primary germ layers. We describe in this paper conditions to induce differentiation of embryonic stem cells reliably and at high efficiency into adipocytes. A prerequisite is to treat early developing embryonic stem cell-derived embryoid bodies with retinoic acid for a precise period of time. Retinoic acid could not be substituted by adipogenic hormones nor by potent activators of peroxisome proliferator-activated receptors. Treatment with retinoic acid resulted in the subsequent appearance of large clusters of mature adipocytes in embryoid body outgrowths. Lipogenic and lipolytic activities as well as high level expression of adipocyte specific genes could be detected in these cultures. Analysis of expression of potential adipogenic genes, such as peroxisome proliferator-activated receptors gamma and delta and CCAAT/enhancer binding protein beta, during differentiation of retinoic acid-treated embryoid bodies has been performed. The temporal pattern of expression of genes encoding these nuclear factors resembled that found during mouse embryogenesis. The differentiation of embryonic stem cells into adipocytes will provide an invaluable model for the characterisation of the role of genes expressed during the adipocyte development programme and for the identification of new adipogenic regulatory genes.
Assuntos
Adipócitos/citologia , Tecido Adiposo/embriologia , Células-Tronco/citologia , Animais , Biomarcadores , Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Dimetil Sulfóxido/farmacologia , Expressão Gênica , Camundongos , Proteínas Nucleares/metabolismo , Fotomicrografia , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Células-Tronco/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tretinoína/farmacologiaRESUMO
Fatty acids and thiazolidinediones act as potent activators of the adipose differentiation program in established preadipose cell lines. In this report, the effects of these agents on the differentiation pathway of myoblasts have been investigated. Exposure of C2C12N myoblasts (a subclone of the C2C12 cell line) to thiazolidinediones or fatty acids prevents the expression of myogenin, alpha-actin, and creatine kinase, thus abolishing the formation of multinucleated myotubes. These treatments lead in parallel to the expression of a typical adipose differentiation program including acquisition of adipocyte morphology and activation of adipose-related genes. A similar transition toward the adipose differentiation pathway also occurs in mouse muscle satellite cells maintained in primary culture. Thiazolidinediones exert their adipogenic effects only in non-terminally differentiated myoblasts; myotubes are insensitive to the compounds. Continuous exposure to inducers after growth arrest is not required to maintain the adipose phenotype, but proliferation of adipose-like C2C12N cells leads to a complete reversion toward undifferentiated cells able to undergo either myogenic or adipogenic differentiation depending on the composition of culture medium. These results indicate that adipogenic inducers, such as thiazolidinediones or fatty acids, specifically convert the differentiation pathway of myoblasts into that of adipoblasts.
