ATF4 Regulates Lactation In Bovine Mammary Epithelial Cells

Activating transcription factor 4(ATF4)is a signal molecule downstream of endoplasmic reticulum-resident kinase(PERK)pathway in three signaling pathways of endoplasmic reticulum stress.ATF4 plays a significant role in many aspects such as individual development,cellular stress response,amino acid secretion,apoptosis and tumorigenesis.At present,there are limited reports about the expression and regulation of ATF4 gene during the development of bovine mammary glands.In this study,the mid-lactating bovine mammary epithelial cells were invoked as a model to explore the relationship between ATF4 gene expression and the development and lactation of bovine mammary glands.The research provided the basic information and the theoretical basis for artificially regulating milk quality and milk yield.In this study,experimental animals were Chinese Holstein bovines.Laser confocal and q RTPCR detection was first used to detect the expression and location of ATF4 in the mammary gland tissues of puberty,lactation and dry periods,so as to identify the expression and location differences of ATF4 in each stage of bovine mammary development.Taking bovine mammary epithelial cells(BMECs)cultured in vitro in mid-lactating bovines as the research object,ATF4 si RNA was transiently transfected into BMECs by using RNA interference technology.After ATF4 gene interference,the relative methods were used to detect mammary epithelial cell viability and cell proliferation.Then detect changes in gene expression of signal pathways related to lactation and in the secretion of triglyceride.Co-immunoprecipitation(co-IP)was used to detect the interaction between ATF4 and the important lactation regulatory protein m TOR.And ATF4 interference and m TOR signaling pathway inhibitors rapamycin co-treated BMECs,then detect the expression of lactation-related signaling pathway genes and amino acid transport carriers like CHAC1 expression etc.and the secretion of triglycerides.In order to further determine the regulatory effect of ATF4 on amino acid transporters,the experiment investigated the interaction between ATF4 and transcription-related regulatory proteins through co-IP.To verify its relationships with transcriptionrelated regulatory proteins,the ATF4 gene of BMECs was silenced and BMECs were treated with amino acid starved,so as to study the expression regulation of ATF4 gene on amino acid transporters.The results of laser confocal detection showed that ATF4 was mainly expressed in the cytoplasm of mammary gland tissues.ATF4 was highly expressed in bovine mammary gland tissues of puberty and lactation,and the expression of mammary tissues in dry milk was low.The results suggested that ATF4 may be associated with the regulation of mammary gland development and lactation.In this study,the effect of ATF4 on milk fat,milk protein synthesis,cell proliferation and apoptosis were further detected.The result showed that PPAR-γ,SREBP-1,β-casein protein expression decreased,Caspase3 protein expression increased,m TOR protein expression difference was not significant.Cell viability is significantly decreased.In addition,the proliferation rate of BMECs by ATF4 gene interference was significantly lower than that of the blank control group and the negative group.And the expression of Cyclin D1 decreased,indicating that ATF4 can participate in the regulation of lactation and BMECs proliferation.In this experiment,Co-IP technology was used to identify the interaction between ATF4 and m TOR.Subsequently,this experiment performed ATF4 gene interference and added rapamycin to treat BMECs.The results showed that when m TOR was inhibited alone,ATF4 protein expression decreased,while PPAR-γ,SREBP-1,β-casein and Cyclin D1 protein expression decreased,and Caspase3 protein expression increased.When m TOR inhibited with ATF4 interfered at the same time,the protein expression of PPAR-γ,SREBP-1,β-casein and Cyclin D1 decreased significantly,and the expression of Caspase3 increased significantly.The above experimental results indicate that m TOR-ATF4 can regulate milk fat,milk protein synthesis,cell proliferation and apoptosis.The result also showed that when ATF4 was inhibited in BMECs and added rapamycin to culture medium,the m RNA expression of amino acid transporters such as CHAC1 in cells were significantly down-regulated.To further determine the mechanism of ATF4 regulated the expression of amino acid transports,the experiment detected that ATF4 interacted with Nuclear Factor erythroid 2-related factor 2(NRF2)by co-IP technology during normal culture to regulate the expression of amino acid transporter.And ATF4 interacted with mammalian deacetylase 6(SIRT6),a protein that regulating homeostasis,during nutrition deficiency culture to decrease the expression of amino acid transporters.However,when the expression of ATF4 rose sharply during the endoplasmic reticulum stress,ATF4 did not interact with SIRT6 or NRF2.At this time ATF4 may performed other biological functions.In summary,the normal lactation and proliferation of BMECs required the regulation of m TORATF4.ATF4 played an important biological role in regulating the expression of amino acid transporters.