Effects And Signal Pathway Of Se-Met On Protein Synthesis In Mouse C2C12 Myoblast Cells

Amino acids are not only substrates of protein translation,but also a class of foreign signal molecules that can activate the phosphatidylinositol 3-kinase（PI3K）and its downstream the mammalian target of rapamycin（m TOR）signaling pathways,thereby increasing protein synthesis in cells.Selenomethionine（Se-Met）is an amino acid containing selenium presented in proteins,and can be used as a kind of feed additive with low toxicity,low pollution and high utilization rate.Annexin A2 is a kind of Ca²⁺-dependent phospholipid binding proteins.Our previous laboratory studies have found that Annexin A2 can sense to the signals of amino acids and activate the downstream PI3K-AKT-m TOR signaling pathways.Protein kinase C（PKC）is a kinase widely distributed in human tissues and cells and can activate Annexin A2 phosphorylation.It is still unknownwhether Se-Met can regulate the PKC-Annexin A2signaling pathway and thereby activate the PI3K-AKT-m TORsignaling and promote protein synthesis in cells.This study aims to reveal the regulatory role of Se-Met in protein synthesis in mouse myoblast C2C12 and its regulation on the above related signaling pathway.In this study,in vitro cultured mouse myoblast C2C12 was used as the research object.Cells were treated with 0,25,50,75,and 100μM Se-Met.Western blotting detected that,with the concentration of Se-Met increasing,the protein expression level of Cyclin D1 gradually increased,reached a peak at 50μM,and then decreased.The MTT analysis detected that,the absorbance at490 nm reached a peak at 50μM.These results suggest that Se-Met regulates the proliferation of C2C12 cells in a dose-dependent manner.Western blotting detected that,with Se-Met concentration increasing,m TOR phosphorylation and the total structural protein level in cells labeled with puromycin were gradually increased,reached a peak at 50μM,then gradually decreased.These results indicated that Se-Met can dose-dependently regulate protein synthesis in C2C12 cells.To uncover the pathway via which Se-Met regulates protein synthesis in C2C12 cells,the PI3K inhibitor-LY 294002 was added to inhibit the PI3K signaling pathway.Western blotting detected that PI3K inhibition almost eliminated the stimulation of Se-Met on m TOR phosphorylation and total structural protein level in cells,indicating that PI3K activation is required for Se-Met to activate m TOR and promote protein synthesis in cells.Cells were treated with 50μM Se-Met and transfected with Annexin A2 si RNA or overexpression Annexin A2,The results showed that Annexin A2 significantly positive regulate the stimulation of Se-Met on PI3K and m TOR phosphorylation and total structural protein level in cells.Our previous Mass spectrometry data indicated that PKCαmight bing to Annexin A2.We further investigated the role of PKCαlocated upstream of Annexin A2 and regulation in Se-Met signaling to Annexin A2.Co-IP（Co-Immunoprecipitation）proved that Annexin A2 interacted with PKCαin C2C12 cells,Adding Se-Met can make the combination between the two stronger.Cells were treated with 50μM Se-Met and transfected with Annexin A2 si RNA.The results showed that PKC knockdown significantly inhibited the up-regulation of phosphorylation of Annexin A2,PI3K and m TOR,and decreased total structural protein in cells.These data suggest that Se-Met regulatesAnnexin A2 phosphorylation in C2C12 cells in a PKCα-dependent manner,and hints that PKCαmight be an upstream kinase of Annexin A2.In summary,Se-Met can dose-dependently regulate the expression of Cyclin D1,phosphorylation of m TOR,and total structural protein synthesis in C2C12 cells.Se-Met regulates these signaling pathways and total structural protein synthesis through the PKCα-Annexin A2-PI3K signaling pathway,thereby affecting C2C12 cell proliferation and protein synthesis.Our findings provide further theoretical and experimental basis for the utilization of Se-Met as a feed additive to promote muscle protein production of livestock and poultry.