Molecular Mechanism Of ERK5 In Promoting PC12 Cell Differentiation

BACKGROUNDThe disorder of neuronal proliferation and differentiation affect the repair and regeneration of traumatic central nervous system after damaged,and further induce a variety of neurological diseases.Previous experimental results demonstrated that PTEN silencing promotes the proliferation and differentiation of PC12,suggesting that PTEN may regulate neural cell differentiation in a certain way.ERK5 also plays an important part in the development of the nervous system.PTEN may interact with ERK5 to ensure normal differentiation of nerve cells.ERK5 is a novel member of the MAPK family discovered in recent years.Compared with other MAPK family members p38,ERK1/2,and JNK1/2/3,ERK5 has highly homologous amino terminus,and the specific carboxyl-terminal transcriptional activation region and nuclear localization sequence result in ERK5 molecular weight being about 2 times that of the classical MAPK family.The amino-terminus of ERK5 includes a kinase domain for binding to the upstream molecule MEK5.ERK5 is commonly expressed in many tissues and is activated by various extracellular stimuli such as cellular stress and growth factors,thus participates in the physiological processes such as cell proliferation,differentiation,and survival,playing a vital role.PC12 cell line,which is rat pheochromocytoma cells,are derived from rat adrenal glands.PC12 cells can adhere to the culture dish through polylysine.PC12 also expresses NGFR(Nerve Growth Factor Receptor),and can be induced to differentiation by NGF.It has the same embryonic origin as neuroblasts and is thought to be immature neurons.This allows PC12 cells to serve as a cell model for neuronal differentiation and neurosecretion,and is typically used in neurological studies.In our study,we have explored the mechanism that ERK5 regulates the proliferation or differentiation of PC12 cells,providing a theoretical basis for clinical application.PURPOSEThe effect of ERK5 on the proliferation of PC12 was established by analyzing the proliferation ability and cell viability of ERK5-overexpressing or ERK5-silencing PC12 cells.After ERK5 overexpression or silencing,and using ERK5 inhibitors,NGF was used to induce differentiation of PC12 cells.The differentiation of PC12 cells was analyzed to determine the differentiation-related molecules involved in the differentiation of ERK5-regulated PC12 cells,Effecting on differentiation of PC12 cells.ERK5 downstream molecules were found and ERK5 was identified to activate someone transcriptional regulators,then developing the signal pathway of ERK5 after NGF stimulation.METHODIn order to improve the efficiency of transfection,PC12 cells infected with ERK5 overexpression,LV5 empty vector control,ERK5 silencing,LV3 empty vector control lentivirus.And the puromycin was used to select positive cells,building stable cell lines.Subsequently,the proliferation of PC12 cells was tested by plotting cell growth curves and measuring CCK-8 absorbance values.NGF was used to induce differentiation of PC12 cells,and Western blot was used to determine the expression level of Marker protein after induction by NGF.Real-time PCR,microscopy,and other methods were used to compare the differentiated Marker mRNA levels and PC12 morphological changes during PC12 cells differentiation.The proportion of differentiated cells was calculated using GraphPad 5.0 to determine neurite extension of PC12 cells after ERK5 overexpression or silencing.The ERK5-specific inhibitor XMD8-92 was utilized to inhibit ERK5 activity and assisted in verifying the differentiation of PC12 in the case where ERK5 activity was inhibited.Real-time PCR was used to compare the expression levels of ERK5 downstream transcriptional regulators in differently treated PC12 cells,and ChIP assay was used to determine whether these downstream genes were involved in the ERK5 signaling pathway.RESULT1.The rate of growth and proliferation of ERK5 silencing PC12 cells was significantly higher than that of PC12 cells without treatment or empty vector control.The cell growth rate of ERK5 overexpression was ERK5 is the opposite2.The expression of Marker-GAP43 protein was significantly increased in ERK5-overexpressing PC12 cells after NGF induction,but the ERK5-silencing PC12 cells showed opposite results.In addition,the obvious changes of cell morphology can be found in cell photographs taken by microscope.ERK5-overexpression PC12 cells are more and longer at the fifth day after NGF induction,the branches of which are significantly more than that of PC12 cells without treatment,so does the expression of GAP43 protein,which is located in cytoplasm.However,the same things are opposite in ERK5-silencing PC12 cells.Real-time PCR was utilized for detecting the expression of Marker in PC12 cells treated with NGF on the 0th,1st,3rd,and 5th day after differentiation.The results showed that the expression of GAP43 and NGFR gradually increased during differentiation.While the expression levels of Nestin and Tubb3 did not change markedly,and there was no statistical difference.However,the expression level of GAP43 and NGFR in the ERK5 overexpression group was significantly higher than that in the non-treatment group PC12 cells.Results of ERK5-silencing PC12 cells were opposite.The consequences of XMD8-92-treated PC12 cells were consistent with the ERK5 silencing group,indicating that the expression of GAP43 decreased after the activity of ERK5 was inhibited.3.The p-ERK5 protein level was significantly decreased at a concentration of 10 ?M and 2 h.However,when the maximum concentration was 20 ?M and the maximum time was 48 h,the protein level of p-ERK5 was the lowest,indicating that XMD8-92 had the best inhibitory effect in a dose-and time-dependent manner.4.The mRNA levels of CREB,FoxO3,and Pml in ERK5-overexpressing PC12 cells were significantly higher than those in untreated PC12 cells,whereas the transcription levels of c-Jun,c-Myc,and Twist1 were significantly lower in ERK5-overexpressing PC12 cells than in untreated PC12 cells.However,results of ERK5-silencing PC12 cells were opposite.5.The ChIP results showed that the p-ERK5 antibody can pull down the DNA fragments of the c-Jun and Pml promoter regions.CONCLUSION1.ERK5 inhibits the proliferation of PC12 cells and promotes the differentiation of PC12 cells.2.ERK5 may promote the differentiation of PC12 cells by promoting the transcription of CREB,FOXO3 and PML and inhibiting the transcription of c-Jun,c-Myc and Twist1.And may be through indirect regulation of c-Jun,PML transcription.