Regulatory Mechanism Of NFκB1 On Milk Synthesis And Proliferation Of Bovine Mammary Epithelial Cells

Milk is the production of physiological activities in the mammary gland of female mammals.It is not only a natural nutrition for feeding calf in the period of growth and development,but also one of the most important necessities for human beings.The regulatory mechanism of milk synthesis is one of the most important and basic research areas in life science.To explore the signaling pathways related to milk synthesis and search for important signaling moleculars will provide valuable experimental evidences for promoting milk production and quality.The regulatory role of gene expression in milk synthesis depends on nutritional and hormonal regulation.Regulation of amino acids on milk protein synthesis has been a hot research area in recent years.At present,some breakthroughs have been achieved on studies of the regulatory roles of amino acids and hormones in regulating milk synthesis,but research on the molecular mechanism of milk synthesis is still not deepgoing.More work need to be done to explore the molecular mechanism of regulation on milk synthesis and to improve the network of molecular regulation.The nuclear factor of κB(NFκB)family has a central role in coordinating a wide variety of genes important for many biological processes including cell growth,apoptosis,immune responses and inflammation,however knowledge of its functions on milk synthesis is rarely reported.In this research,we reveal that NFκB1 senses amino acids(methionine)and hormones(estrogen)via PI3 K signaling pathway and positively regulates milk synthesis and proliferation of bovine mammary epithelial cells(BMECs)by promoting the expressions of its target genes(m TOR,SREBP-1c,β4Gal-T2 and Cyclin D1).This study also confirms that glycyl t RNA synthetase(Gly RS)mediates methionine(Met)and estrogen(E)stimulation,and promotes NFκB1 phosphorylation.This research both enriches and improves the network of molecular regulation on milk synthesis.It also provides important experimental basis on the mechanism of milk synthesis regulated by nutrients and hormones.In this research,BMECs were cultured and purified by tissue culture method.The expressions of Cytokeratin 18 and β-casein were detected to identify the purity and lactating function of BMECs,respectively.In this study,two lactating cell models were established by adding Met(0.6 mmol/L)or E(2.72×10-2 μg/L)to the culture medium.q RT-PCR and Western blot analysis showed that NFκB1 expression was obviously upregulated,which suggests that NFκB1 is involved in milk synthesis.Immunofluorescence observation was applied to detect the expression and subcellular localization of NFκB1 and p-NFκB1.From the observations,we found that Met and E enhanced the expression of NFκB1 and triggered its nuclear translocation as well as phosphorylation.These results suggest that NFκB1 might mediate environmental stimuli and promote milk synthesis in BMECs.We further exploited gene function studies including NFκB1 overexpression and silencing methods.Results showed that NFκB1 positively regulated milk synthesis and cell proliferation by promoting the expressions of mTOR,SREBP-1c,β4Gal-T2 and Cyclin D1.Further results also showed that NFκB1 overexpression promoted the secretion of triglyceride and lactose as well as proliferation of BMECs.Interfering NFκB1 reflected an opposite result.In addition,detection of BODIPY-labeled lipid droplet accumulation by laser confocal microscope showed that NFκB1 promoted the synthesis of lipid droplet in BMECs.Our data reveal that NFκB1 positively regulates milk synthesis and proliferation of BMECs.To explore the mechanism through which mediates milk synthesis and proliferation of BMECs,we first predicted the consensus κB binding sites in the gene promoters of m TOR,SREBP-1c,β4Gal-T2 and Cyclin D1.The analytical result showed that all of these genes might contain the κB binding sites in their promoters.Ch IP-PCR analysis verified the binding of NFκB1 to these gene promoters.Ch IP-q PCR assays confirmed that the binding of NFκB1 to these gene promoters was obviously promoted after Met or E stimulation.These findings reveal that Met and E trigger NFκB1 activation,enhance the binding of NFκB1 to target gene promoters and regulate the expressions of these genes in milk synthesis and cell proliferation at transcriptional level.To detect the mechanism through which NFκB1 senses environmental stimuli such as Met or E,wortmannin and rapamycin were used as the inhibitors of PI3 K and m TOR,respectively.Inhibiting PI3 K markedly suppressed the protein levels of NFκB1 and p-NFκB1,however,inhibiting mTOR showed no obvious effect.Further study was accessed to determine whether PI3 K mediates the activation of NFκB1.After adding Met or E together with PI3 K inhibitor,the stimulation of Met or E on the expression of NFκB1 was obviously weakened.Data confirm that amino acids and hormones activate NFκB1 via PI3 K signaling pathway.Furthermore,we explored the mechanism through which GlyRS mediates Met and E to promote NFκB1 phosphorylation.We detected both of the effects of Gly RS overexpression together with silencing NFκB1 on the expression of NFκB1 target genes and the effects of Met or E stimulation together with silencing Gly RS on NFκB1 expression and phosphorylation.Results suggest that Gly RS mediates Met or E to promote NFκB1 phosphorylation and regulates m TOR,SREBP-1c,Cyclin D1 signaling pathways mainly via NFκB1.We also explored the effects of silencing Gly RS together with silencing GCN2 on NFκB1 expression and phosphorylation.Results reveal that the effect of Gly RS on promoting NFκB1 phosphorylation is independent of GCN2 signaling pathway and NFκB1 expression.Recent research in our lab has confirmed that Gly RS directly interacts with NFκB1 and promotes its phosphorylation showing a consistent result with our conclusions.Taken together,our study reveals that NFκB1 is a critical regulator in milk synthesis and proliferation of BMECs.Met and E activate NFκB1 via PI3 K and Gly RS signaling pathways.p-NFκB1 regulates the expressions of mTOR,SREBP-1c,β4Gal-T2 and Cyclin D1 at transcriptional level by binding to their promoters.