Study On The Role Of Epac In Proliferation And Growth Hormone Synthesis And Secretion Of Rat GH3 Cells And The Interaction Between Epac And Extracellular Regulated Protein Kinase Cell Signaling Pathway

Part 1:The role of PKA and Epac in mediating cAMP induced proliferation of GH3 cellsObjective To verify the effect of cAMP on the proliferation of rat anterior pituitary GH3 cells,and to explore the role of cAMP's downstream effectors,PKA and Epac,in mediating this effect.Methods CCK8 method was used to study the effects of Forskolin,PKA and Epac's inhibitors on the proliferation of GH3 cells.Then,Epac specific activator 8-pCPT-2'-O-Me-cAMP was applied to GH3 cells.The effect of Epac activation on the proliferation of GH3 cells was confirmed by CCK8 and EDU at the cellular and DNA levels.Results Forskolin promotes the proliferation of GH3 cells.PKA and Epac's inhibitors can both partially inhibit this stimulatory effect,but the GH3 cell proliferation is still statistically significant.8-pCPT-2'-O-Me-cAMP specifically activates Epac and causes GH3 cells proliferation,it works in a concentration-dependent manner.Conclusion cAMP induced proliferation of GH3 cells could be mediated by both PKA and Epac.Activation of Epac alone can also stimulate GH3 cells proliferation.Part 2:Activation of Epac promoted expression levels of the phosphorylated ERK1/2 in GH3 cell lines but had no effect on CREB phosphorylationObjective To study the effect of cAMP on ERK1/2 and CREB in GH3 cells and its relationship with Epac,to explore the downstream signal transduction pathway of cAMP-Epac.Methods The phosphorylation levels of ERK1/2 and CREB were detected by western blot after GH3 cells were treated with Forskolin,PKA and Epacinhibitors,B-Raf inhibitor,Epac activator or Epac SiRNA in a proper manner.Results cAMP significantly promoted p-ERK1/2 expression level of GH3 cells,this promotion was partially inhibited by PKA or EPAC inhibitors.cAMP significantly promoted the expression of p-CREB in GH3 cells,which could only be inhibited by PKA inhibitor but not by Epac inhibitor.Specific activation of Epac also promoted p-ERK1/2 expression in GH3 cells,and drug blocking of B-Raf could inhibit this effect.Conclusion In GH3 cells,cAMP cross talks with MAPK/ERK signal pathway,and stimulates p-ERK1/2 expression,this crosstalk can be mediated by both PKA and Epac The effect of cAMP on p-CREB expression was not associated with Epac.Crosstalk between cAMP-Epac and MAPK/ERK requires B-Raf.Part3:The role of B-Raf and MAPK/ERK signaling pathway in Epac activation induced GH3 cells proliferationObjective To study the role of B-Raf and MAPK/ERK signaling pathway in Epac activation induced GH3 cells proliferation.Methods The proliferation rate of GH3 cells was studied with CCK8 test,after cells were treated with B-Raf and ERK 1/2 inhibitor,Epac activator in a proper manner.Western blot was used to detect the expression of cyclin D3.Results Epac activation induced GH3 cells proliferation could be absolutely prevented by B-Raf or ERK 1/2 inhibitor,Epac activation induced stimulation of cyclinD3 expression could also be prevented by B-Raf or ERK 1/2 inhibitorConclusion Epac activation induces GH3 cells proliferation through the activation of ERK 1/2,and requires B-Raf.Part 4:The Effects of Epac activation on growth hormone synthesis and secretion of GH3 CellsObjective To study the effects of Epac activation on growth hormone synthesis and secretion of GH3 cells.Methods After GH3 cells were treated with Epac activator for indicated time,growth hormone mRNA level was detected by real-time RT-PCR,intracellular and extracellular growth hormone levels were detected by ELISA.Results The growth hormone mRNA level,intracellular and extracellular growth hormone levels had no difference between Epac activator treated and untreated GH3 cells.Conclusion Epac activation has no significant effect on growth hormone synthesis and secretion of GH3 cells.