RESUMO
DNA methylation is an epigenetic event involved in regulation of gene transcription during cell differentiation. DNA methyltransferases (DNMT) play a role in differentiation of neural stem cells into neurons. The aim of this study was to determine whether nerve growth factor (NGF) was involved in differentiation of mouse hippocampal neuronal cell line (HT22) as assessed by IncuCyte. Quantitative PCR and western blot were used to measure gene and protein expression of DNMT as well as the activity of DNMTs. Treatment with NGF was found to upregulate both gene and protein expressions as well as total activity of DNMTs in differentiating HT22 cells. Compared to undifferentiating cells, the percentage of differentiating cells at S phase increased significantly when incubated with NGF. In undifferentiated cells, NGF failed to induce gene and protein expressions and activity of DNMTs. Data demonstrate that differentiation of HT22 cells by exposure to NGF involve the activation of DNMTs pathway.
Assuntos
Diferenciação Celular/genética , Hipocampo/fisiologia , Fator de Crescimento Neural/genética , Neurônios/fisiologia , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Camundongos , Fator de Crescimento Neural/metabolismoRESUMO
BACKGROUND: Learning and memory is a complex process. Some reports have shown that protein kinases (PKs) and phosphatases (PPs) are important mediators in this process. And it is also well known that protein serine/threonine phosphatase 1 (PP1) and DNA methylation are critically involved in learning and memory. METHODS: In the current study, the mice and cultured cells (NG108-15) were treated with vehicle or 5-Aza-2'-deoxycytidine (5-aza-cdR), a DNA methyltransferase (DNMT) inhibitor. The ability of learning and memory of mice was detected by Morris Water Maze, while the mRNA and protein expression levels of DNMTs and PP1γ in mice hippocampus were measured by real-time PCR and western-blot. To further clarify whether the 5-aza-cdR effects on learning and memory depend on cell proliferation/apoptosis or not, the effects of 5-aza-cdR on the cell proliferation, apoptosis, and PP1γ transcriptional activity were analyzed by using the xCelligence system, flow cytometer and Luciferase reporter assay, respectively. RESULTS: The ability of learning and memory was increased while the expressions of DNMTs and PP1γ were decreased in the hippocampus of mice which were injected with 5-aza-cdR. In vitro experiments showed 10µM 5-aza-cdR inhibited cell proliferation, decreased PP1γ transcription without inducing apoptosis. CONCLUSION: Our data demonstrate that the 5-aza-cdR restrains the expression of PP1γ which is related to learning and memory in the mice.
