RESUMO
Skeletal muscle mass is negatively regulated by several TGF-ß superfamily members. Myostatin (MSTN) is the most prominent negative regulator of muscle mass. Recent studies show that in addition to MSTN, GDF11, which shares a high sequence identity with MSTN, induces muscle atrophy in vitro and in vivo at supraphysiological levels, whereas controversy regarding its roles exists. Furthermore, higher circulating GDF11 levels associate with frailty in humans. On the other hand, little is known about the effect of pathophysiological levels of GDF11 on muscle atrophy. Here we seek to determine whether pathophysiological levels of GDF11 are sufficient to activate Smad2/Smad3 signaling and induce muscle atrophy using human iPSC-derived myocytes (hiPSC myocytes). We first show that incubating hiPSC myocytes with pathophysiological concentrations of GDF11 significantly reduces myocyte diameters. We next demonstrate that pathophysiological levels of GDF11 are sufficient to activate Smad2/3 signaling. Finally, we show that pathophysiological levels of GDF11 are capable of inducing the expression of Atrogin-1, an atrophy-promoting E3 ubiquitin ligase and that FOXO1 blockage reverses the GDF11-induced Atrogin-1 expression and atrophic phenotype. Collectively, our results suggest that GDF11 induces skeletal muscle atrophy at the pathophysiological levels through the GDF11-FOXO1 axis.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miostatina , Humanos , Miostatina/genética , Miostatina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/metabolismo , Fatores de Diferenciação de Crescimento/farmacologia , Atrofia Muscular/patologia , Músculo Esquelético/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células Musculares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína Smad3/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Proteína Smad2/genéticaRESUMO
Rab is a small GTP-binding protein family that regulates various pathways of vesicular transport. Although more than 60 Rab proteins are targeted to specific organelles in mammalian cells, the mechanisms underlying the specificity of Rab proteins for the respective organelles remain unknown. In this study, we reconstituted the Golgi targeting of Rab6A in streptolysin O (SLO)-permeabilized HeLa cells in a cytosol-dependent manner and investigated the biochemical requirements of targeting. Golgi-targeting assays identified Bicaudal-D (BICD)2, which is reportedly involved in the dynein-mediated transport of mRNAs during oogenesis and embryogenesis in Drosophila, as a cytosolic factor for the Golgi targeting of Rab6A in SLO-permeabilized HeLa cells. Subsequent immunofluorescence analyses indicated decreased amounts of the GTP-bound active form of Rab6 in BICD2-knockdown cells. In addition, fluorescence recovery after photobleaching (FRAP) analyses revealed that overexpression of the C-terminal region of BICD2 decreased the exchange rate of GFP-Rab6A between the Golgi membrane and the cytosol. Collectively, these results indicated that BICD2 facilitates the binding of Rab6A to the Golgi by stabilizing its GTP-bound form. Moreover, several analyses of vesicular transport demonstrated that Rab6A and BICD2 play crucial roles in Golgi tubule fusion with the endoplasmic reticulum (ER) in brefeldin A (BFA)-treated cells, indicating that BICD2 is involved in coat protein I (COPI)-independent Golgi-to-ER retrograde vesicular transport.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Brefeldina A/farmacologia , Complexo I de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/genética , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/fisiologia , Complexo de Golgi/genética , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Phosphatidylinositol-3-phosphate (PI3P) is a lipid that is enriched specifically in early endosomes. Given that early endosomes containing PI3P act as a microdomain to recruit proteins that contain a PI3P-binding domain (FYVE domain), the equilibrium between the production and degradation of PI3P influences a variety of processes, including endocytosis and signal transduction via endosomes. In the study reported herein, we have developed a novel analytical method to quantify the amount of PI3P in endosomes by introducing a GST-2xFYVE protein probe into semi-intact cells. The GST-2xFYVE probe was targeted specifically to intracellular PI3P-containing endosomes, which retained their small punctate structure, and allowed the semi-quantitative measurement of intracellular PI3P. Using the method, we found that treatment of HeLa cells with H(2)O(2) decreased the amount of PI3P in endosomes in a p38 MAPK-dependent manner. In addition, H(2)O(2) treatment delayed transport through various endocytic pathways, especially post-early endosome transport; the retrograde transport of cholera toxin was especially dependent on the amount of PI3P in endosomes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
