Study On FLCN Regulating Mammary Epithelial Cell Proliferation And Milk Protein Synthesis In Dairy Cows

Mammary gland development and lactation are strictly regulated by various signaling pathways.Revealing and elucidating lactation-related signaling pathways and mammary gland metabolic regulation networks are important research contents in the field of lactation biology,providing a theoretical basis for improving milk quality and milk production.Folliculin(FLCN)gene as a tumor suppressor gene in various metabolic pathways and cellular processes,and plays an important role in cell adhesion,apoptosis,metabolism,signal transduction,protein synthesis and mitochondrial biosynthesis.However,the expression and function of FLCN in the mammary gland of dairy cows have not been reported.In this study,we aimed to elucidate the molecular mechanism of FLCN that regulates cell proliferation and milk protein synthesis inmammary gland of dairy cows by analyzing cell metabolism,proliferation,apoptosis and signaling functions and interacting proteins.The experiments first used laser confocal to detectthe expression of FLCN in the mammary gland of puberty,lactation and dry milk,in order to identify the expression and differences of FLCN in the mammary gland development of dairy cows.In order to determine the effect of FLCN on the proliferation and lactation of mammary gland epithelial cells in dairy cows,this experiment used a model of cow mammary epithelial cells cultured in vitro.Interference and overexpression of FLCN gene,and detection the expression of AMPK,p-AMPK,mTOR,p-mTOR,Cyclin Dl,Caspase3 and P-casein by qRT-PCR and Western blot,detection cell proliferation using flow cytometry.The interaction between FLCN and AMP activated protein kinase(AMPK)was studied by Co-IP and mass spectrometry.In order to further determine the regulation of FLCN on AMPK signaling pathway,this study overexpressed and interfered with FLCN gene in dairy mammary epithelial cells,and added AMPK activators and inhibitors in culture medium,and detected the expression ofFLCN、AMPK、p-AMPK、mTOR、p-mTOR、Cyclin Dland β-caseinby Western blot.In order to explore the molecular mechanism of FLCN regulating breast energy metabolism through AMPK signaling pathway,the mammary gland epithelial cells were treated by sugar-free medium,andwere used to detect the expression of FLCN、AMPK、p-AMPK、mTOR、p-mTOR、Cyclin D1、ACC and β-casein in glucose-free culture conditions by qRT-PCR and Western blotThe results of laser confocal microscopy showed that FLCN was mainly localized in mammary epithelial cells,and the expression of breast tissue in puberty and lactation was high and dry milk stage was low,indicating that FLCN may be involved in the regulation of mammary gland development and lactation in dairy cows.This experiment further examined the effects of FLCN overexpression and inhibition on milk protein synthesis,cell proliferation and apoptosis.The results showed that FLCN overexpression can up-regulate the protein expression levels of mTOR and β-casein and promote the phosphorylation of mTOR,and the inhibition results are opposite to the overexpression.The cell viability assay showed that overexpression of FLCN resulted in a significant increase in cell number and a significant increase in cell viability.In addition,the proliferation rate of mammary epithelial cells overexpressing FLCN was significantly higher than that of the blank control group and the transfected empty vector group,and the percentage of cells in the S phase and G2-M phase was significantly increased,while the percentage of cells in the G0-G1 phase was significantly decreased.Inhibition experiments are the opposite of overexpression.Overexpression and inhibition of FLCN experiments also showed that FLCN positively regulates the expression of cyclin Cyclin D1,and the results of flow cytometry indicate that FLCN promotes cell proliferation.In addition,the results showed that FLCN negatively regulates the expression of the apoptotic protein Caspase3,and found that FLCN negatively regulates the expression level of AMPK and inhibits its phosphorylation.The above experimental results indicate that FLCN positively regulates the phosphorylation level of mTOR,thereby promoting milk protein synthesis and cell proliferation,inhibiting apoptosis,and negatively regulating AMPK.In this study,the interaction between FLCN and AMPK was studied by Co-IP and mass spectrometry.We found FLCN and FINP1 were detected when AMPK antibody was used for Co-IP.Meanwhile,AMPK and FLCN proteins were detected when FINP1 antibody was used for Co-IP.The results showedFLCN and its binding protein FINP1 can interacted with AMPK,suggesting that FLCN was involved in the regulation of AMPK signaling pathway.To further determine the regulation of FLCN on the AMPK signaling pathway,we overexpressed and interfered with the FLCN gene,and added AMPK activators and inhibitors to the culture medium,respectively.The results showed that the expressionlevels of FLCN,Cyclin D1 andβ-casein were increased,the expressionlevel of mTOR and phosphorylation were decreased when AMPK was activated.The opposite results were obtained when AMPK activity was inhibited.The results showed that AMPK positively regulates the expression of FLCN,negatively regulates the expression and activity of mTOR,and inhibits milk protein synthesis and cell proliferation.Combined with previous studies,FLCN overexpression inhibits the expression and phosphorylation of AMPK,and AMPK activation promotes the expression of FLCN.which forming a negative feedback effect between AMPK and FLCN.The results showed that AMPK positively regulates the expression of FLCN,negatively regulates the expression and activity of mTOR,and inhibits milk protein synthesis and cell proliferation.Consistently,the overexpression of FLCN inhibits the expression and phosphorylation of AMPK,and the activation of AMPK promotes the expression of FLCN,thus forming a negative feedback effect between AMPK and FLCN.We next examined whether FLCN is required in the mammary gland energy metabolism through AMPK signaling pathway.We found that the expression levels of AMPK and FLCN was significantly increased under the condition of glucose-free culture,the phosphorylation of AMPK was significantly increased,while the expression levels of mTOR and its phosphorylation were decreased,the expression levels of β-casein and the gene and protein of ACC(milk fat synthesis related enzyme)was decreased.The results indicated the cellular energy receptor AMPK was activated and the expression level of FLCN was increased to dynamically regulate AMPK activity to inhibit apoptosis,thereby maintaining early cell survival under energy-deficient conditions.Consistently,FLCN participates in the regulation of energy metabolism in mammary gland epithelial cells through the AMPK signaling pathway.In summary,FLCN promotes milk protein synthesis and cell proliferation,and inhibits apoptosis.FLCN is involved in the regulation of AMPK signaling pathway in mammary epithelial cells,and there is a negative feedback relationship between FLCN and AMPK.