Gene Function Of Cullin5 In The Regulation Of Taurine On Milk Synthesis Of Mouse Mammary Epithelial Cells

Milk is an essential nutrition source for the newborn mammals,and also an important food for people of different ages.Milk has high nutritional and economic value.In mammary gland,mammary epithelial cells can synthesize nutrients in the blood into milk,thus the quality and quantity of milk are determined by the proliferation of mammary epithelial cells and their ability to synthesize milk.The mammalian target of rapamycin(mTOR)can respond to amino acids,hormones and other signal molecules,positively regulating cell proliferation and the anabolism of protein,lipid,etc.Taurine(Tau)has been found to enhance cellular antioxidant activity,inhibit apoptosis,and promote protein and lipid anabolism,but the molecular mechanism of its regulatory effect is not clear.The ubiquitinase Cullin5(Cul5)can play an important biological role by regulating a variety of signal pathways,but it has not been reported whether Cul5 can regulate proliferation and milk synthesis of mammary epithelial cells.It’s also largely unknown the function and molecular mechanism of Cul5 in regulating the mTOR signal pathway.This study aims to uncover the effect of Cul5 on proliferation and milk synthesis of mammary epithelial cells and the corresponding molecular mechanism,and whether Cul5 can respond to the signal of Tau to stimulate mTOR activation.In this study,we found that the expression of Cul5 was significantly higher in mammary tissues in lactation than that in puberty or involution stages,suggesting that Cul5 might be related to milk synthesis.Detection of the expression of β-casein in HC11 cells(a mouse mammary epithelial cell line)by immunofluorescence observation showed that HC11 cells could express β-casein,suggesting that HC11 cells has the ability to synthesize milk components.To explore the effect of Cul5 on the proliferation of HC11 cells and the synthesis of milk protein and milk fat,the model of Cul5 gene knockdown and gene activation was constructed using siRNA transfection and CRISPR-d Cas9 technology,respectively.The cell proliferation ability,β-casein protein abundance and triglyceride(TG)content were detected using the EdU and CCK-8 detection kit,Western blotting and a TG content detection kit,respectively.The results showed that the above indexes were decreased by Cul5 knockdown,whereas Cul5 gene activation had opposite effects.These above results demonstrate that Cul5 is a positive regulator of proliferation and milk protein and milk fat synthesis of HC11 cells.The mRNA level and protein phosphorylation level of mTOR were also detected after knockdown and gene activation of Cul5.The results demonstrate that Cul5 positively regulates the mRNA expression and protein phosphorylation(activation)of mTOR,thus promoting cell proliferation and milk protein and fat synthesis.To determine whether Tau affects the proliferation of HC11 cells and the expression of Cul5,different concentrations of Tau(0.48,0.60,0.72,0.84,0.96 m M)were added to the cells.It was found that the ability of cell proliferation,the level of Cul5 mRNA and the abundance of Cul5 protein gradually increased with the increase of Tau concentration.These indexes reached a peak at 0.72 m M,and then gradually decreased.After inhibiting PI3K(phosphatidylinositol 3-kinase)with LY 294002,the stimulation of Tau on the expression of Cul5 protein was almost completely blocked.The subcellular localization experiment of Cul5 by immunofluorescence observation showed that Cul5 was only localized to the nucleus.The above results suggest that Tau promotes proliferation of HC11 cells in a dose-dependent manner,and 0.72 m M was the optimal concentration;Cul5 is localized to the nucleus of HC11 cells;PI3K mediates the stimulation of Tau on Cul5 expression.To further explore whether Tau can stimulate mTOR activation through affecting the expression of Cul5,HC11 cells were treated with Tau together with a Cul5 siRNA.It was found that the promotion of Tau on mRNA expression and protein phosphorylation(activation)of mTOR was almost completely eliminated by Cul5 knockdown.These results demonstrate that Cul5 mediates the promotion of Tau on mRNA expression and protein phosphorylation of mTOR.In conclusion,Tau positively regulates the expression of Cul5 in HC11 cells.Cul5 is a positive regulator of HC11 cell proliferation and milk synthesis,and mediates Tau to promote the expression of mTOR mRNA and the phosphorylation of mTOR protein.Our findings reveal the role and molecular mechanism of Cul5 in milk synthesis and cell proliferation of mammary epithelial cells,and provides a new theoretical basis for the application of taurine to improve milk quality and yield in mammals.