The Role And Molecular Mechanism Of ARID1A In Amino Acid-Stimulated Milk Synthesis In Bovine Mammary Epithelial Cells

Milk,which contains milk protein,milk fat,vitamins and other nutritional components,is an agricultural product with high economic value.Milk production of mammary gland is mainly determined by the milk synthesis and proliferation abilities of mammary epithelial cells(MECs).Revealing the regulation mechanismof milk synthesis in MECs will help to learn more on the lactation gene regulation network,and will provide a basic theoretical basis for the establishment of new mammary gland development and lactation regulation technology.It has been reported that amino acids are one of the most important regulators of mammalian lactation,which can activate the mechanistic target of rapamycin(mTOR),the key signaling molecule for milk synthesis and proliferation,to promote milk synthesis in MECs,but the specific mechanismneeds for further research.AT-rich interaction domain 1A(ARID1A)is a member of the chromatin remodeling complex Switch/sucrose non-fermenable(SWI/SNF),which can regulate cell proliferation,differentiation and apoptosis,affect lipid homeostasis and play a variety of biological functions.However,whether ARID1 A is involved in regulating milk synthesis and responding to amino acid signals has not been reported.This study will reveal the role and molecular mechanismof ARID1 A in amino acid-stimulated milk synthesis in bovine mammary epithelial cells(BMECs).The results will provide a novel theoretical basis for the application of amino acids in milk production.This study found that the protein abundance of ARID1 A was low er in mouse mammary gland tissues of lactating period than that of puberty or involution period.However,mTOR phosphorylation was significantly higher in tissues of lactating period than that of puberty or involution period.These data indicate that ARID1 A might play a key negative role in the regulation of milk synthesis in mammary gland.BMECs were cultured by the digestion method.The purity of BMECs was verified by the immunofluorescence observation of CK18 and β-casein expression in cells.These two proteins were expressed in almost all the cells observed,showing that the purity of BMECs was almost 100%.BMECs were used as the research object,in order to explore whether ARID1 A is a key regulator of milk synthesis,the effects of ARID1 A knockdown and gene activation were detected by using Western blotting,BODIPY staining and triglyceride(TG)content detection.It was found that ARID1 A knockdown significantly increased β-casein protein expression,secretion of TG,and formation of lipid droplets in cells.Cell counting and CCK8 assay detected that ARID1 A knockdown promoted proliferation of BMECs.After ARID1 A gene activation in cells,the level ofβ-casein protein,secretion of TG,formation of lipid droplets,and cell proliferation were all significantly decreased.These above data demonstrate that ARID1 A plays a negative regulatory role in milk synthesis in and proliferation of BMECs.In order to study the effect of ARID1 A on mTOR gene expression and protein phosphorylation,Western blotting and RT-q PCR were used to detect the protein expression and mRNA levels of mTOR after ARID1 A knockdown and gene activation.ARID1 A knockdown significantly increased mTOR phosphorylation and the expression of mTOR mRNA,whereas ARID1 A gene activation had the opposite effect.These data suggest that ARID1 A negatively regulates mRNA expression and subsequent protein activation of mTOR.To further explore the mechanismvia which ARID1 A inhibits mTOR gene expression,the binding sites of ARID1 A and three modified histones(H3K27ac,H3K4me3,and H3K27me3)in the mTOR promoter were identified by Ch IP-PCR.Western blotting combined with Ch IP-q PCR were exploited to analyze the effect of ARID1 A gene activation and knockdown on the binding of histone modifications in the mTOR promoter.The protein levels of H3K4me3,H3K27me3 and H3K27 ac remained unchanged regardless of ARID1 A knockdown or gene activation.Interestingly,Ch IP-PCR analysis detected that ARID1 A and these modified histones were all merely bound to the-548～-793 nt sequence in the-1 ～-2000 nt region of the mTOR promoter.Next,Ch IP-q PCR analysis detected that ARID1 A knockdown significantly increased the binding of H3K27 ac to the mTOR promoter,whereas ARID1 A gene activation had the opposite effect.Both ARID1 A knockdown and gene activation had no effect on the binding of H3K4me3 and H3K27me3 to the mTOR promoter.These data suggest that ARID1 A inhibits mTOR transcriptional activation through decreasing the deposition of H3K27 ac on the mTOR promoter.In order to further study whether ARID1 A plays a role in the regulation of milk synthesis by amino acids,0.6 mM Met or 0.6 mM Leu was added together with gene activation of ARID1 A.The effects of ARID1 A gene activation on mTOR gene expression,milk synthesis and cell proliferation stimulated by Met and Leu were analyzed by Western blotting,RT-q PCR,BODIPY staining,TG detection and CCK8.ARID1 A gene activation almost totally blocked the stimulation of mTOR phosphorylation and mTOR mRNA expression stimulated by Met and Leu.In addition,ARID1 A gene activation almost totally blocked the stimulation on β-casein protein expression,TG secretion,lipid droplet formation and cell proliferation stimulated by Met and Leu.These data suggest that ARID1 A is a key negative regulator in the stimulation of Met and Leu on mTOR gene transcription,milk synthesis and cell proliferation.The above study found that the protein level of ARID1 A decreased significantly after the addition of Met and Leu.To investigate the corresponding molecular mechanism,Western blotting and RT-q PCR were used to detect the protein and mRNA levels of ARID1 A after the addition of Met and Leu.It was found that the addition of Met and Leu reduced the protein level of ARID1 A but did not change its mRNA level.It is suggested that the regulation of ARID1 A by Met and Leu is not related to the regulation of gene expression.To test this hypothesis,cells were treated with Met or Leu for 24 h as well as CHX(10 μg/mL)for 0,2,4,6 h,and the protein levels of ARID1 A were detected by Western blotting.The protein synthesis inhibitor CHX did not affect the protein level of ARID1 A in BMECs at 0,2,4,6 h after CHX treatment,which was different fromthe Met or Leu treatment group.Amino acids were added to cells along with 50 n M autophagy inhibitor chloroquine(CQ)or 10 μM proteasome inhibitor MG132,the protein level of ARID1 A was detected by Western blotting.The addition of Met and Leu decreased ARID1 A protein level,however,the autophagy inhibitor CQ did not restore the down-regulation of ARID1 A protein expression by Met and Leu,whereas the proteasome inhibitor MG132 totally rescued the protein level of ARID1 A downregulated by Met and Leu.Additionally,Met and Leu treatments significantly increased the level of ubiquitinated ARID1 A protein.In summary,these above results reveal that Met and Leu induce the degradation of ARID1 A through the ubiquitin-proteasome system.To further explore the molecular mechanismvia which Met and Leu induce ARID1 A degradation.In our experimental results of mass spectrometry analysis of the protein interactome of ARID1 A in BMECs,we noted that the E3 ubiquitin ligase tripartite motif containing 21(TRIM21)might be associated with ARID1 A ubiquitination.TRIM21 exhibited expression not only in the cytoplasmbut also in the nucleus by Western blotting analysis.Co-IP analysis detected the interaction between TRIM21 and ARID1 A.In order to study whether TRIM21 is a key mediator of ARID1 A degradation induced by Met and Leu,cells were transfected with a TIRM21 si RNA and treated with Met(0.6 mM)or Leu(0.6 mM)for 24 h.The protein levels of TRIM21 and ARID1 A were detected by Western blotting.Western blotting showed that TRIM21 knockdown significantly increased the expression level of ARID1 A protein.Met and Leu stimulated the expression of TRIM21,furthermore,TRIM21 knockdown almost totally abolished ARID1 A degradation induced by Met and Leu.These above data demonstrate that TRIM21 is an E3 ubiquitin ligase for ARID1 A ubiquitination and a key mediator in Met and Leu signaling to ARID1 A degradation in BMECs.In summary,current study demonstrates that ARID1 A is a key negative regulator of gene transcription of mTOR,milk synthesis,and cell proliferation stimulated by Met and Leu,through decreasing H3K27 ac deposition on the mTOR promoter.Met and Leu induce ARID1 A ubiquitination and degradation,which is mediated by TRIM21.Our findings uncover that Met and Leu promote mTOR gene transcription for milk synthesis and cell proliferation through the TRIM21-ARID1 A signaling pathway.