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

Mammalian mammary tissue secretes milk to provide nutrition and immune protection for the growth of the next generation.Milk as a natural nutrient is also an essential food for human life.The main ingredients in milk are very complex,including milk protein,milk fat,lactose and so on.The signal pathways associated with milk synthesis,the key signal molecules regulating milk synthesis and their mechanism of action are important contents of animal husbandry and life science research.PURB is a member of the Pur protein family.The Purr family protein can form isomorphic and heterotypic complexes,and specifically binds purin-rich SS DNA(sense or antisense)or RNA with high affinity.They preferentially combined with a repeat sequence of GGN(N not G).In our previous study,it was found that the expression of PURB in bovine mammary epithelial cells(BMECs)was increased significantly when cells were stimulated by methionine,suggesting that PURB as a transcription factor may regulate the synthesis of BMECs milk.At present,there are few studies on the function and mechanism of PURB in the world.There is no research to reveal whether PURB plays a role in BMEC milk synthesis.In this study,we aim to reveal the role and molecular mechanism of PURB in amino acid-stimulated milk prtein and milk fat synthesis in BMECs.In this study,BMECs were isolated by tissue mass culture method,purified and passed down for subsequent studies.Cell morphology and growth rate were observed under an inverted microscope.Expression of Cytokeratin 18 and β-casein were detected by immunofluorescence(IF)to identify cell purity and lactation function.The expression levels of α-casein and β-casein were significantly increased by the addition of 0.6 mmol/L Met and 0.6mmol /L Leu.The results of lipid drop test and triglyceride test showed that Met and Leu significantly promote the synthesis of milk fat in cells.From the above data,we have successfully established the lactation model of cells.,qRT-PCR and western blotting indicated that the expression levels of PURB protein and PURB mRNA were significantly increased by the addition of 0.6mmol /L Met and 0.6mmol /L Leu.Immunofluorescence observation showed that PURB localization was in the nucleus,and Met and Leu stimulation enhanced PURB localization.The results showed that the Met and Leu significantly promoted the expression of PURB in BMECs,suggesting that PURB may be an important regulator of milk synthesis stimulated by the Met and Leu.The PURB gene was overexpressed and interfered with a recombinant plasmid and a siRNA.When the PURB gene was over-expressed,the expression of PURB,α-casein,and β-casein were increased significantly,the synthesis of triglyceride and formation of lipid droplets were increased significantly,and the levels of mTOR phosphorylation and SREBP-1c proteins were also increased significantly.When the PURB gene was knocked down,the expression of PURB,α-casein,and β-casein were significantly reduced,triglyceride secretion and the synthesis of lipid droplets were significantly reduced,and the levels of mTOR phosphorylation and SREBP-1c proteins were also significantly reduced.The above research results revealed that PURB has the effect of positively regulating mTOR and SRBP-1c signal molecules to promote milk protein and milk fat synthesis.To explore the molecular mechanism through which PURB responses to amino acids such as Met and Leu to regulate milk synthesis,wortmannin was applied to suppress the PI3 K activity.Western blotting analysis detected that PI3 K inhibition completely blocked the promotion of two amino acids on PURB expression.These results showed that Met and Leu promoted the expression of PURB protein through the PI3 K pathway.To further explore whether the PURB mediates the mTOR and SREBP-1c signal molecules that are regulated by Met and Leu,PURB siRNA was transferred simultaneously into cells when the cells were stimulated with amino acids(Met and Leu).Western blotting analysis detected that PI3 K inhibition completely blocked the promotion of two amino acids on mTOR phosphorylation and SREBP-1c protein levels.The results revealed that PURB mediates Met and Leu stimulation on mTOR and SREBP-1c signal molecules.ChIP-PCR and ChIP-qPCR were used to identify the sites where PURB binds to the promoters(-1 ~-2 000 bp)of mTOR and SRBP-1c genes.It was found that there was a binding site of PURB in the promoter of mTOR or SRBP-1c gene.ChIP-qPCR analysis further detected that Met and Leu stimuli promoted the binding of PURB to these two target gene promoters.PURB gene over-expression and knockdown also revealed that PURB positively regulates mRNA levels of mTOR and SREBP-1c.These experimental results show that Met and Leu stimulate PURB expression and promote the binding PURB to mTOR,SREBP-1c gene promoters to initiate gene transcription.Several lines of studies pointed that PURB plays a critical role in gene transcription,but there were fewer reports in the worldwide by contrast with many other transcription factors.In our study,PURB was first identified in BMECs as a positive regulator of milk protein and milk fat synthesis.PURB was located in the nucleus,and its expression was promoted by amino acids(Met and Leu)via the PI3 K signaling pathway.PURB can bind to the mTOR and SREBP-1c gene promoters,and Met and Leu stimulate this binding.We also found that PURB mediates the stimulation of the Met and Leu on the mTOR and SREBP-1c signaling pathways leading to milk protein and fat synthesis.In summary,through systematic and comprehensive research,this experiment revealed that PURB promotes mTOR and SREBP-1c signaling pathways under the stimulation of exogenous amino acids,and positively regulates milk protein and milk fat synthesis.PURB binds to the promoter specific sequence of mTOR and SREBP-1c,and positively regulates these downstream target genes at the transcription level.This study also revealed that the PURB protein is one of the key regulatory factors in the milk synthesis of BMECs induced by amino acids.