RESUMEN
Lindbladione (1) is a neural stem cell differentiation activator isolated from Lindbladia tubulina by our group. Hes1 dimerization inhibitory activity of lindbladione (1) was discovered using our original fluorescent Hes1 dimer microplate assay. We also found that lindbladione (1) accelerates the differentiation of neural stem cells. We conducted the first total synthesis of lindbladione (1) via Heck reaction of 1-hexene-3-one 7 with iodinated naphthoquinone 12, which was provided by Friedel-Crafts acylation followed by Claisen condensation, in the presence of Pd (II) acetate. Careful deprotection of the benzyl groups of 13 successively provided lindbladione (1). Synthesized lindbladione (1) exhibited potent Hes1 dimer inhibition (IC50 of 2.7 µM) in our previously developed fluorescent Hes1 dimer microplate assay. Synthesized lindbladione (1) also accelerated the differentiation of C17.2 mouse neural stem cells into neurons dose dependently, increasing the number of neurons by 59% (2.5 µM) and 112% (10 µM) compared to the control. These activities are comparable to those of naturally occurring lindbladione (1) isolated from L. tublina.
Asunto(s)
Amoeba/química , Naftoquinonas/síntesis química , Células-Madre Neurales/citología , Factor de Transcripción HES-1/química , Factor de Transcripción HES-1/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Ratones , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Naftoquinonas/química , Naftoquinonas/farmacología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Conformación Proteica , Multimerización de Proteína/efectos de los fármacosRESUMEN
The processes of cell proliferation and differentiation are intimately linked during embryogenesis, and the superfamily of (basic) Helix-Loop-Helix (bHLH) transcription factors play critical roles in these events. For example, neuronal differentiation is promoted by class II bHLH proneural proteins such as Ngn2 and Ascl1, while class VI Hes proteins act to restrain differentiation and promote progenitor maintenance. We have previously described multi-site phosphorylation as a key regulator of tissue specific class II bHLH proteins in all three embryonic germ layers, and this enables coordination of differentiation with the cell cycle. Hes1 homologues also show analogous conserved proline directed kinase sites. Here we have used formation of Xenopus primary neurons to investigate the effects of xHes1 multi-site phosphorylation on both endogenous and ectopic proneural protein-induced neurogenesis. We find that xHes1 is phosphorylated in vivo, and preventing phosphorylation on three conserved SP/TP sites in the N terminus of the protein enhances xHes1 protein stability and repressor activity. Mechanistically, compared to wild-type xHes1, phospho-mutant xHes1 exhibits greater repression of Ngn2 transcription as well as producing a greater reduction in Ngn2 protein stability and chromatin binding. We propose that cell cycle dependent phosphorylation of class VI Hes proteins may act alongside similar regulation of class II bHLH proneural proteins to co-ordinate their activity.
Asunto(s)
Neurogénesis , Factor de Transcripción HES-1/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neuronas/citología , Neuronas/metabolismo , Fosforilación , Estabilidad Proteica , Factor de Transcripción HES-1/química , Proteínas de Xenopus/química , Xenopus laevis/metabolismoRESUMEN
OBJECTIVES: Neural stem cells (NSCs) are self-renewing, undifferentiated and multipotent precursors that can generate neuronal and glial lineages. MicroRNAs (miRNAs) are small non-coding RNAs that act crucial roles in cell proliferation, differentiation and migration. However, the role of miR-1297 in the development of NSCs is still unknown. MATERIALS AND METHODS: Primary NSCs were isolated from rat's embryos. The expression of miR-1297 and Hes1 were measured by qRT-PCR. Western blot was performed to detect the protein expression of Hes1, ß-tubulin-III and GFAP. RESULTS: We showed that miR-1297 expression was upregulated during NSC differentiation, while the expression of Hes1 was decreased during NSC differentiation. Elevated expression of miR-1297 promoted the NSCs viability and increased the formation of NSCs to neurospheres. Ecoptic expression of miR-1297 promoted ß-tubulin-III expression in the NSCs. Overexpression of miR-1297 decreased GFAP expression in the NSCs. Furthermore, we demonstrated that miR-1297 regulated NSCs viability and differentiation by directly targeting Hes1. Overexpression of miR-1297 suppressed Hes1 expression in the NSCs. CONCLUSIONS: These results suggested that miR-1297 played an important role in NSCs viability and differentiation through inhibiting Hes1 expression.
Asunto(s)
MicroARNs/metabolismo , Células-Madre Neurales/metabolismo , Factor de Transcripción HES-1/metabolismo , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Regulación hacia Abajo , Embrión de Mamíferos/citología , Inmunohistoquímica , MicroARNs/genética , Células-Madre Neurales/citología , Ratas , Alineación de Secuencia , Factor de Transcripción HES-1/química , Factor de Transcripción HES-1/genética , Tubulina (Proteína)/metabolismo , Regulación hacia ArribaRESUMEN
A diabetes susceptibility gene, immunoglobulin-like domain containing receptor 2 (Ildr2), encodes a transmembrane protein localized to the endoplasmic reticulum membrane that is closely related to hepatic lipid metabolism. The livers of ob/ob mice in which Ildr2 is transiently overexpressed are relieved of hepatic steatosis. However, the molecular mechanisms through which ILDR2 affects these changes in hepatic lipid metabolism remain unknown. This study aimed to identify ILDR2-interacting proteins to further elucidate the molecular mechanisms underlying the role of ILDR2 in lipid homeostasis. We purified ILDR2-containing protein complexes using tandem affinity purification tagging and identified ZNF70, a member of the Kruppel C2H2-type zinc finger protein family, as a novel ILDR2-interacting protein. We demonstrated that ZNF70 interacts with ZFP64 and activates HES1 transcription by binding to the HES1 promoter. In addition, HES1 gene expression is increased in ILDR2-knockdown HepG2 cells, in which ZNF70 is translocated from the cytoplasm to the nucleus, suggesting that ZNF70 migration to the nucleus after dissociating from the ILDR2-ZNF70 complex activates HES1 transcription. These results support a novel link between ILDR2 and HES1 gene expression and suggest that ILDR2 is involved in a novel pathway in hepatic steatosis.