Molecular Mechanism Via ARID4B Mediating The Stimulation Of Taurine On Protein Synthesis And Proliferation Of Myoblast C2C12

Amino acids are a kind of exogenous signal molecules,which can activate the phosphatidylinositol 3-kinase(PI3K),the mechanistic target of rapamycin(m TOR)and other signal pathways for cell proliferation and protein synthesis.Taurine(Tau)is a β-sulfamic acid,the most abundant free amino acid in the body,and found in very high concentrations in muscle cells.Previous reports have shown that Tau has physiological functions including regulating calcium ion levels,maintaining osmotic pressure balance and improving the body’s antioxidant capacity,etc.Tau can be used as a feed additive for animal husbandry,but it is still unknown the effects and underlying molecular mechanism of Tau on protein synthesis and proliferation of muscle cells.AT-rich interaction domain 4B(ARID4B)is a newly discovered transcription factor,which plays an important role in cell growth and differentiation,but it has not been reported the role of and corresponding molecular mechanism of ARID4 B in protein synthesis and cell proliferation.Therefore,this study intends to reveal the molecular mechanism via which ARID4 B mediates the regulation of Tau on protein synthesis and proliferation of muscle cells.In this study,mouse myoblast C2C12 was used as the research object.Tau(0,60,120,180,240μM)was added into the culture medium of C2C12 cells,the CCK-8 assay kit was used for the detection of cell number,the results showed that,with the increase of Tau concentration,the cell number gradually increased,reached the highest at 120 μM,and then gradually decreased;the SUn SET non-radioactive method was used for the detection of the protein synthesis rate in cells,and BCA(bicinchoninic acid)protein quantification kit detection was used for the detection of total protein content,the results showed that,with the increase of Tau concentration,the protein synthesis rate and total protein content gradually increased,reached a peak at 120 μM,and then gradually decreased.Western blotting and q RT-PCR analysis detected that Tau(120 μM)significantly promoted m TOR phosphorylation,ARID4 B protein and m RNA levels.These above results demonstrate that Tau can regulate C2C12 cell proliferation and protein synthesis in a dose-dependent manner,and excessive Tau might have inhibitory effects.The effects of ARID4 B on the proliferation and protein synthesis of C2C12 cells were further explored.After treatment with Tau at the optimum concentration(120 μM),AKT phosphorylation and ARID4 B protein level were significantly increased.Addition of PI3 K inhibitor LY 294002 completely blocked the promotion of Tau on AKT phosphorylation and ARID4 B expression.q RT-PCR results showed that PI3 K inhibition completely blocked the promoting effect of Tau on ARID4 B m RNA expression.These results suggest that PI3 K is a key signal molecule that mediates Tau regulation of ARID4 B expression.Cells were treated with Tau(120 μM)and transfected with an ARID4 B si RNA,the results showed that ARID4 B knockdown blocked the stimulation of Tau on cell number,protein synthesis rate and total protein content,as well as the promotion of Tau on m TOR phosphorylation and m TOR m RNA level.The CRISPR-d Cas9 method was used to activate ARID4 B gene expression.The results showed that ARID4 B gene activation increased the number of C2C12 cells,protein synthesis rate,total protein content,m TOR phosphorylation level and m TOR m RNA level.These above data demonstrate that ARID4 B is a key mediator for Tau to promote C2C12 cell proliferation,protein synthesis and m TOR m RNA expression.Chromatin immunoprecipitation(Ch IP)was used to identify the possible binding site of ARID4 B on the m TOR gene promoter(-1～-5000 bp).Ch IP-PCR detected that all of ARID4 B,H3K27ac and H3K27me3 bound to the-4591～-4368 bp site in the m TOR gene promoter,and Ch IP-q PCR further detected that Tau stimulated ARID4 B binding to this site.Western blotting detected that ARID4 B knockdown and gene activation did not affect the protein levels of H3K27 ac and H3K27me3 in cells.Ch IP-q PCR detected that ARID4 B knockdown or gene activation did not affect H3K27me3 binding to the m TOR gene promoter,but increased or decreased H3K27 ac binding,respectively.Collectively,these data demonstrate that Tau promotes ARID4 B binding to the m TOR gene promoter,thereby triggers the binding of H3K27 ac to this promoter and activate m TOR gene transcription.In conclusion,Tau regulates C2C12 cell proliferation and protein synthesis in a dose-dependent manner;ARID4B is a key mediator for Tau to promote C2C12 cell proliferation,protein synthesis and m TOR m RNA expression.This study reveals the molecular mechanism of ARID4B-mediated Tau regulation of m TOR gene transcription to promote protein synthesis and proliferation of muscle cells,providing a theoretical basis for Tau application in muscle growth and protein production of livestock and poultry.