The Role Of GSK3? In Goat Skeletal Muscle Satellite Cells Proliferation And Differentiation

Glycogen synthase kinase 3(GSK3)is a ubiquitous serine/threonine kinase in mammalian,and it has various directed or undirected substrates performing vital functions in many cell signaling pathways.GSK3 has two mammalian isoforms:GSK3? and GSK3?.Some studies show that GSK3? is involved in cell proliferation and differentiation.The purpose of this study is to explore the role of GSK3? in goat SMSCs proliferation and differentiation by adding GSK3? inhibitor of SB216763.Besides,six different fragments of the MyHC2a promoter were successfully constructed and the transcriptional regulation of GSK3? on MyHC2a promoter was explored by double luciferase reporter assay system,which provide a theoretical basis for the further study of the role of GSK3? in muscle growth and development.The main results are as follows:1.When SMSCs began to grow,the cells presented with long spindle-shaped.On the 1 d of differentiation,the cells grew in the same direction and fused with each other to form plenty of myotubes at 7 d.The qPCR results showed that on SMSCs proliferation period,Pax7 and MyoD were expressed with a high level,while MyHC and MyoG hardly expressed.After entering differentiation stage,the expression of Pax7 was significantly decreased(P<0.01).MyoD maintained a high level(3 d),then reduced significantly in 5 d and 7 d(P<0.05).Compared with the proliferation stage,the expression of MyHC was significantly up-regulated at 3 d,5 d and 7 d(P<0.01),and has no significant difference at I d(P>0.05),and the expression level of MyoG gene was significantly higher during SMSCs differentiation than 0 d(P<0.01),and reached the highest at 3 d(P<0.01),then decreased significantly at 5 d and 7 d(P<0.01).2.SB216763 inhibited the activity of GSK3?,which inhibited the differentiation of goat skeletal muscle satellite cells,and down-regulated the expression of differentiation marker genes.Compared with the control group,the expression of Pax7 in SB216763 treated group was not significantly different in the proliferative phase,while the expression of MyoD gene was significantly decreased at 0 d(P<0.05).The MyHC expression level was significantly down-regulated at 5 d and 7 d(P<0.01),and was no significant difference between the two groups after 0 d,1 d and 3 d differentiation.The expression of MyoG gene did not change significantly in the proliferation period,and the expression level of MyoG gene decreased significantly at differentiation stage(P<0.01).3.The promoter fragment activity of MyHC2a was detected by double luciferase reporter system.The results showed that the activities of the six promoter fragments of MyHC2a gene were significantly higher than that of pGL3-basic(P<0.05)in the SMSCs proliferation stage.The luciferase activity of-154/+55 was significantly lower than that of fragment-229/+55(P<0.05),indicating the presence of a positive regulatory element between fragment-154 to-229.The luciferase activity of fragment-229/+55 was significantly higher than that of fragment-466/+55(P<0.05),and the fragments-466/+55,-514/+55,-704/+55 and-1038//+55 were not significant difference,which indicated that there was a negative regulatory element between the fragment-229 and the fragment-466.In the SMSCs differentiation phase,the luciferase activity of the fragment-466/+55 was significantly lower than that of the fragment-514/+55(P<0.05),indicating that there was a positive regulatory element between fragments of-466 to-514;The luciferase activity of-704/+55 was significantly lower than that of fragment-514/+55(P<0.05),which indicated that there was a negative control element between the fragment-514 and the fragment-704.And The luciferase activity of-704/+55 was significantly higher than that of fragment-1038//+55(P<0.05),which indicated that there was a negative control element between the fragment-704 and-1038.4.The activity of MyHC2a promoter was detected by inhibition of GSK3? by double luciferase reporter system.The results showed that GSK3? inhibition increased the activities of MyHC2a promoter fragment-466/+55,-514/+55,-704/+55 and-1038/+55(P<0.05),and down-regulated the activity of fragment-229/+55(P<0.05),compared with the activity of six promoter fragments on the phase of SMSCs.Moreover,-466/+55 and-1038/+55 were significantly promoted(P<0.05)on the differentiation stage of SMSCs,while the activities of-229/+55 and-514/+55 were significantly down-regulated(P<0.05).