ABSTRACT
Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and size of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)-dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , DNA-Binding Proteins/metabolism , Embryonic Stem Cells/metabolism , Neurons/metabolism , Phosphoproteins/metabolism , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Base Sequence , Brain/embryology , Brain/physiology , Cell Cycle Proteins , DNA-Binding Proteins/genetics , Embryo, Mammalian/cytology , Embryonic Stem Cells/cytology , Female , Gene Expression Regulation, Developmental , Mice, Inbred Strains , Molecular Sequence Data , Muscle Proteins/genetics , Neurons/cytology , Phosphoproteins/genetics , Pregnancy , Signal Transduction , TEA Domain Transcription Factors , Trans-Activators , Transcription Factors/genetics , YAP-Signaling ProteinsABSTRACT
Creatine is a nitrogenous organic acid known to function in adenosine triphosphate (ATP) metabolism. Recent evidence indicates that creatine regulates the differentiation of mesenchymal stem cells (MSCs) in processes such as osteogenesis and myogenesis. In this study, we show that creatine also has a negative regulatory effect on fat cell formation. Creatine inhibits the accumulation of cytoplasmic triglycerides in a dose-dependent manner irrespective of the adipogenic cell models used, including a C3H10T1/2 MSC line, 3T3-L1 preadipocytes, and primary human MSCs. Consistently, a dramatic reduction in mRNA expression of adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), glucose transporters, 1 and 4 (Glut1, Glut4), and adipocyte markers, aP2 and adipsin, was observed in the presence of creatine. Creatine appears to exert its inhibitory effects on adipogenesis during early differentiation, but not late differentiation, or proliferation stages through inhibition of the PI3K-Akt-PPARγ signaling pathway. In an in vivo model, administration of creatine into mice resulted in body mass increase without fat accumulation. In summary, our results indicate that creatine downregulates adipogenesis through inhibition of phosphatidylinositol 3-kinase (PI3K) activation and imply the potent therapeutic value of creatine in treating obesity and obesity-related metabolic disorders.
Subject(s)
Adipogenesis , Creatine/pharmacology , Down-Regulation , Mesenchymal Stem Cells/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , 3T3 Cells , Animals , CCAAT-Enhancer-Binding Protein-alpha/genetics , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Cells, Cultured , Complement Factor D/genetics , Complement Factor D/metabolism , Fatty Acid-Binding Proteins/genetics , Fatty Acid-Binding Proteins/metabolism , Glucose Transporter Type 1/genetics , Glucose Transporter Type 1/metabolism , Glucose Transporter Type 4/genetics , Glucose Transporter Type 4/metabolism , Humans , Insulin/pharmacology , Male , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , PPAR gamma/genetics , PPAR gamma/metabolism , Triglycerides/metabolismABSTRACT
Notch has a broad range of regulatory functions in many developmental processes, including hematopoiesis, neurogenesis, and angiogenesis. Notch has several key functional regions such as the RBP-Jκ/CBF1 association module (RAM) domain, nuclear localization signals (NLS), and ankyrin (ANK) repeats. However, previous reports assessing the level of importance of these domains in the Notch signaling pathway are controversial. In this study, we have assessed the level of contribution of each Notch domain to the regulation of mammalian neural stem cells in vivo as well as in vitro. Reporter assays and real-time polymerase chain reactions show that the ANK repeats and RAM domain are indispensable to the transactivation of Notch target genes, whereas a nuclear export signal (NES)-fused Notch intracellular domain (NICD) mutant defective in nuclear localization exerts a level of activity comparable to unmodified NICD. Transactivational ability appears to be tightly coupled to Notch functions during brain development. Unlike ANK repeats and RAM domain deletion mutants, NES-NICD recapitulates NICD features such as promotion of astrogenesis at the expense of neurogenesis in vitro and enhancement of neural stem cell character in vivo. Our data support the previous observation that intranuclear localization is not essential to the oncogenesis of Notch1 in certain types of cells and imply the importance of the noncanonical Notch signaling pathway in the regulation of mammalian neural stem cells.