RESUMO
Excessive fat accumulation is a major risk factor for the development of type 2 diabetes mellitus and other common conditions, including cardiovascular disease and certain types of cancer. Here, we identify a mechanism that regulates adiposity based on the activator of autophagy TP53INP2. We report that TP53INP2 is a negative regulator of adipogenesis in human and mouse preadipocytes. In keeping with this, TP53INP2 ablation in mice caused enhanced adiposity, which was characterized by greater cellularity of subcutaneous adipose tissue and increased expression of master adipogenic genes. TP53INP2 modulates adipogenesis through autophagy-dependent sequestration of GSK3ß into late endosomes. GSK3ß sequestration was also dependent on ESCRT activity. As a result, TP53INP2 promotes greater ß-catenin levels and induces the transcriptional activity of TCF/LEF transcription factors. These results demonstrate a link between autophagy, sequestration of GSK3ß into late endosomes and inhibition of adipogenesis in vivo.
Assuntos
Adipócitos/enzimologia , Adipogenia , Tecido Adiposo/enzimologia , Adiposidade , Autofagia , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Nucleares/metabolismo , beta Catenina/metabolismo , Células 3T3-L1 , Adipócitos/patologia , Tecido Adiposo/patologia , Adulto , Animais , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/enzimologia , Feminino , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Hiperplasia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Transporte Proteico , Espanha , Suécia , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Fatores de Tempo , Ativação Transcricional , Via de Sinalização Wnt , beta Catenina/genéticaRESUMO
The precise regulation of extravillous trophoblast invasion of the uterine wall is a key process in successful pregnancies. Kisspeptin (KP) has been shown to inhibit cancer cell metastasis and placental trophoblast cell migration. In this study primary cultures of first trimester human trophoblast cells have been utilized in order to study the regulation of invasion and angiogenesis-related genes by KP. Trophoblast cells were isolated from first trimester placenta and their identity was confirmed by immunostaining for cytokeratin-7. Real-time quantitative RT-PCR demonstrated that primary trophoblast cells express higher levels of GPR54 (KP receptor) and KP mRNA than the trophoblast cell line HTR8Svneo. Furthermore, trophoblast cells also expressed higher GPR54 and KP protein levels. Treating primary trophoblast cells with KP induced ERK1/2 phosphorylation, while co-treating the cells with a KP antagonist almost completely blocked the activation of ERK1/2 and demonstrated that KP through its cognate GPR54 receptor can activate ERK1/2 in trophoblast cells. KP reduced the migratory capability of trophoblast cells in a scratch-migration assay. Real-time quantitative RT-PCR demonstrated that KP treatment reduced the expression of matrix metalloproteinase 1, 2, 3, 7, 9, 10, 14 and VEGF-A, and increased the expression of tissue inhibitors of metalloproteinases 1 and 3. These results suggest that KP can inhibit first trimester trophoblast cells invasion via inhibition of cell migration and down regulation of the metalloproteinase system and VEGF-A.
Assuntos
Movimento Celular , Implantação do Embrião , Regulação da Expressão Gênica/efeitos dos fármacos , Kisspeptinas/farmacologia , Neovascularização Fisiológica , Primeiro Trimestre da Gravidez , Trofoblastos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Implantação do Embrião/efeitos dos fármacos , Implantação do Embrião/genética , Feminino , Humanos , Kisspeptinas/antagonistas & inibidores , Kisspeptinas/genética , Kisspeptinas/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Gravidez , Primeiro Trimestre da Gravidez/efeitos dos fármacos , Primeiro Trimestre da Gravidez/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Kisspeptina-1 , Trofoblastos/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Mammalian DOR was discovered as a gene whose expression is misregulated in muscle of Zucker diabetic rats. Because no DOR loss-of-function mammalian models are available, we analyze here the in vivo function of DOR by studying flies mutant for Drosophila DOR (dDOR). RESULTS: We show that dDOR is a novel coactivator of ecdysone receptor (EcR) that is needed during metamorphosis. dDOR binds EcR and is required for maximal EcR transcriptional activity. In the absence of dDOR, flies display a number of ecdysone loss-of-function phenotypes such as impaired spiracle eversion, impaired salivary gland degradation, and pupal lethality. Furthermore, dDOR knockout flies are lean. We find that dDOR expression is inhibited by insulin signaling via FOXO. CONCLUSION: This work uncovers dDOR as a novel EcR coactivator. It also establishes a mutual antagonistic relationship between ecdysone and insulin signaling in the fly fat body. Furthermore, because ecdysone signaling inhibits insulin signaling in the fat body, this also uncovers a feed-forward mechanism whereby ecdysone potentiates its own signaling via dDOR.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Ecdisona/metabolismo , Regulação da Expressão Gênica/fisiologia , Insulina/metabolismo , Receptores de Esteroides/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Corpo Adiposo/metabolismo , Componentes do Gene , Regulação da Expressão Gênica/genética , Técnicas de Inativação de Genes , Metamorfose Biológica/fisiologia , Modelos Biológicos , Mutação/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The insulin/TOR signaling pathway plays a crucial role in animal homeostasis, sensing nutrient status to regulate organismal growth and metabolism. We identify here the Drosophila B' regulatory subunit of PP2A (PP2A-B') as a novel, conserved component of the insulin pathway that specifically targets the PP2A holoenzyme to dephosphorylate S6K. PP2A-B' knockout flies have elevated S6K phosphorylation and exhibit phenotypes typical of elevated insulin signaling such as reduced total body triglycerides and reduced longevity. We show that PP2A-B' interacts with S6K both physically and genetically. The human homolog of PP2A-B', PPP2R5C, also counteracts S6K1 phosphorylation, indicating a conserved mechanism in mammals. Since S6K affects development of cancer and metabolic disease, our data identify PPP2R5C as a novel factor of potential medical relevance.
Assuntos
Proteínas de Drosophila/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Animais , Drosophila/metabolismo , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Técnicas de Inativação de Genes , Humanos , Insulina/metabolismo , Longevidade , Fosfoproteínas Fosfatases/deficiência , Fosfoproteínas Fosfatases/genética , Fosforilação , Transdução de Sinais , Triglicerídeos/metabolismoRESUMO
The regulation of autophagy in metazoans is only partly understood, and there is a need to identify the proteins that control this process. The diabetes- and obesity-regulated gene (DOR), a recently reported nuclear cofactor of thyroid hormone receptors, is expressed abundantly in metabolically active tissues such as muscle. Here, we show that DOR shuttles between the nucleus and the cytoplasm, depending on cellular stress conditions, and re-localizes to autophagosomes on autophagy activation. We demonstrate that DOR interacts physically with autophagic proteins Golgi-associated ATPase enhancer of 16 kDa (GATE16) and microtubule-associated protein 1A/1B-light chain 3. Gain-of-function and loss-of-function studies indicate that DOR stimulates autophagosome formation and accelerates the degradation of stable proteins. CG11347, the DOR Drosophila homologue, has been predicted to interact with the Drosophila Atg8 homologues, which suggests functional conservation in autophagy. Flies lacking CG11347 show reduced autophagy in the fat body during pupal development. All together, our data indicate that DOR regulates autophagosome formation and protein degradation in mammalian and Drosophila cells.