Molecular Mechanisms Of 14-3-3β Regulating Milk Proteins Synthesis In BMECs

Proliferation and lactation of bovine mammary epithelial cells are regulated by a variety of signal networks.Research on important functional genes related to lactation and their regulatory networks has always been the focus of lactation biology.Therefore,this part of research will provide theoretical basis for improving milk quality and milk yield.As a class of adaptor proteins,14-3-3 proteins are key regulatory components of many important cellular processes,such as signal transduction,protein folding and degradation and cell cycle.Previous laboratory studies on RNA-seq transcriptome sequencing of mammary tissue of dairy cows showed that the expression of 14-3-3β gene was significantly increased during peak lactation period compared with that in late lactation period.At the same time,study has shown that mice fibroblast cell line-NIH3T3,the 14-3-3β and Insulin-like growth factor I receptor(Insulin –like growth factor I receptor,IGFIR).IGFIR signaling pathway plays an important role in mammary development and lactation regulation of bovine mammary gland,suggesting that 14-3-3β may be involved in the regulation of mammary gland lactation.Research also found that prior to the lab in lactation bovine mammary epithelial cells,and activation transcription factor 4(Activatingtranscriptionfactor4,ATF4)interact with 14-3-3β in stress signaling pathway.Therefore,it is speculated that 14-3-3β may be involved in stress signaling transduction of bovine mammary epithelial cells.In this study,14-3-3β was selected as the research object to reveal the molecular mechanisms of 14-3-3β involved in regulating the proliferation,milk protein synthesis and cellular stress of bovine mammary epithelial cells.First,this study determined the role of 14-3-3β in cell proliferation and milk protein synthesis in bovine mammary epithelial cells.The 14-3-3β interacting proteins were analyzed by mass spectrometry and Co-IP,and the interaction between 14-3-3β and IGFIR was verified.After inhibition of 14-3-3β gene expression by RNAi,IGFIR and expressions of its phosphorylated proteins,p-AKT1,p-m TOR,Cyclin D1,milk fat and β-casein were detected,and cell proliferation and cell viability were detected.After 14-3-3β gene inhibition,IGF-I or leucine was added to promote lactation performance.m RNA and protein expressions of lactation related genes in IGFIR signaling pathway were detected by q RT-PCR and WB.The results showed that 14-3-3β interacted with IGFIR.Inhibition of 14-3-3β gene expression significantly reduced cell proliferation ability and viability,decreased the expressions of lactation related protei ns in IGFIR signaling pathway,and decreased the expression of cyclin D1 protein.Compared with the supplementation of IGF-1 or leucine to promote lactation performance,the supplementation of IGF-I or leucine after 14-3-3βgene inhibition significantly reduced the m RNA and protein expression of lactation related genes in the IGFIR signaling pathway,and the lactation effect of IGF-I or leucine was weakened.These results suggested that 14-3-3β can play a positive role in regulating lactation effect through IGF-I and leucine mediated by IGFIR signaling pathway.This study further analyzed the regulatory effect of 14-3-3β on endoplasmic reticulum stress and cell stress induced by amino acid starvation in bovine mammary epithelial cells.Co-IP and mass spectrometry were used to identify and analyze the interactions between 14-3-3β and eukaryotic translation initiator factor 2α(e IF2α)and ATF4;The m RNA and protein expressions of 14-3-3β,e IF2α and ATF4 in mammary tissues of adolescent and lactation cows were detected by q RT-PCR and WB.The expressions of stress related proteins such as p-e IF2α,ATF4 and CHOP were detected after inhibition of 14-3-3β gene expression.After inhibition of 14-3-3β gene expression,endoplasmic reticulum stress response was induced by Thapsigargin(TG)or cell stress induced by amino acid starvation.The m RNA and protein expressions of stress-related genes,such as e IF2α,ATF4 and CHOP,were detected by q RT-PCR and WB.The results showed that 14-3-3β interacted with e IF2α and ATF4,and the expressions of stress-related proteins such as p-e IF2α and ATF4 decreased after 14-3-3β gene inhibition.Compared with single application of TG or amino acid starvation to induce stress response,the m RNA expressions of stress signaling pathway relate d genes was up-regulated,while the protein expressions was significantly decreased after the inhibition of 14-3-3β gene.And the stress response induced by TG or amino acid starvation was significantly reduced.The results showed that 14-3-3β was involved in regulating the normal stress response of bovine mammary epithelial cells by interacting with e IF2α.In conclusion,14-3-3β plays a positive role in regulating lactation effect through IGF-I and Leu mediated by IGFIR signaling pathway.By interacting with e IF2α,14-3-3β is a necessary condition for cellular stress response,and is involved in regulating cellular stress signaling transduction and protein translation,thus indirectly regulating lactation.Milk protein synthesis is regulated by a series of transduction pathways,including the IGFIR-m TOR mechanism and cell stress response.In the presence of 14-3-3β,these pathways can accurately regulate milk protein synthesis to ensure normal lactation.The interrelationship between these signaling pathways highlights the complexity of the regulation of milk protein synthesis,and the coordination and interaction between anabolism and catabolism can maintain protein homeostasis.This study fully revealed the molecular mechanisms of 14-3-3β involved in the regulation of milk protein synthesis and cell proliferation in mammary epithelial cells,and provided a new experimental idea and research direction for the regulation of dairy mammary gland lactation.