Role Of UFL1 In LPS-Induced Inflammatory Responses And Milk Protein And Fat Synthesis In Bovine Mammary Epithelial Cells

Mastitis is the most frequent,costly and complex disease of dairy cows,which poses a great threat to the milk production and quality and brings huge economic loss.Bovine mammary epithelial cells with a typical characteristic of milk synthesis and secretion,can bind LPS to TLR4 and activate NF-?B,leading to a sequential transcriptional regulatory cascades and resulting in inflammation and cell damage.UFL1 was reported to function as a UFM1 E3 ligase in ufmylation(Ufml-mediated conjugation).It has recently been identified by independent studies as an important regulator of NF-?B signaling,cellular stress response,and is necessary for the proper synthesis of enzymes and secretory function of the exocrine glands.But little is known about its function in bovine mammary epithelial cells.Therefore,the objective of this project is to study the physiological role and underlying mechanism of UFL1 in LPS-induced inflammatory response and lactation in order to find a new approach to develop anti-inflammatory medicine for sub-and clinical mastitis and to breed mastitis resistance dairy cattle.The results of this study showed that:1.UFL1 suppresses the production of inflammatory mediators in LPS-stimulated bovine mammary epithelial cells.Mammary epithelium,the predominant cell type in the mammary gland,can bind LPS to pathogen-associated molecular pattern molecules via activating various PRRs,e.g.,TLRs and NLRs.Generally,the binding of LPS to a TLRs or NLRs can lead to a sequential transcriptional regulatory cascades,cause upregulation of inflammatory cytokines such as IL-1?,TNF-?,and IL-6,resulting in inflammation.UFL1,an UFM1 E3 ligase,has been recently identified as a significant regulator of several signaling pathways.It's a protein with many functions.In this study,we investigated the modulating Effects of UFL1 on the regulation of LPS-induced inflammation.We found that the the mRNA and protein expression levels of UFL1 in bovine mammary gland tissue and BMECs were significantly increased after LPS treatment.UFL1 knockdown resulted in a significant further increase in levels of TNF-?,IL-6,IL-1? and NO in LPS-challenged BMECs.Conversely,compared with the Con+LPS group,overexpression of UFL1 inhibited the expression of LPS-induced flammatory cytokines and NO production.These results suggest that UFL1 could effectively alleviate LPS-induced inflammation via regulating secretion of inflammatory cytokines(TNF-?,IL-1? and IL-6)and NO in bovine mammary epithelial cells.2.UFL1 alleviates LPS-induced cell damage in bovine mammary epithelial cells.Although UFL1 was originally characterized as an UFM1 E3 ligase that plays essential roles in protein ufmylation,its function seems to be more diverse.It can also act as a vital regulator of cellular stress response.In this study,we investigated the modulating Effects of UFL1 on the regulation of LPS-induced cell damage,with a focus on apoptosis,ER stress,autophagy,pyroptosis and ROS.The results showed that UFL1 depletion aggravated the LPS-induced cell damage by exacerbating LPS-induced apoptosis(increased the expression of Bax/Bcl-2 and activity of caspase-3),ER stress(increased the expression of CHOP,HSP70,and GRP78),autophagy(increased LC3-II and decreased P62 expression),pyroptosis(increased NLRP3 expression,Caspase-1 activation and IL-1?secretion)and ROS production.Overexpression of UFL1 relieved the LPS-induced ER stress,apoptosis,autophagy,pyroptosis and ROS production,thereby alleviating the LPS-induced cell damage.Collectively,UFL1 could alleviate LPS-induced cell damage in bovine mammary epithelial cells and thereby act as a potential therapeutic target for bovine mastitis.3.UFL1 regulates milk protein and fat synthesis of bovine mammary epithelial cells via the mTOR signaling pathway.UFL1 mainly located in the endoplasmic reticulum which plays essential roles in fatty acid metabolism,cell proliferation,synthesis and secretion of enzymes in exocrine glands.Yet its physiological function in milk synthesis and proliferation of bovine mammary epithelial cells remains unknown.In this study,we investigated the role of UFL1 in cell proliferation and milk synthesis,with a particular emphasis on protein and fat synthesis,and then further investigated the effects of UFL1 on milk synthesis in LPS-induced BMECs.We found that UFL1 silence negatively regulated the proliferation of BMECs,as indicated by the decrease in cell viability and cells in G2 and S phase,whereas UFL1 overexpression positively regulated proliferation of BMECs,as indicated by the increase in cell viability and BMECs in G2 and S phase.Our gene function studies showed that UFL1 knockdown decreased the level of WAP,ELF5,CSN2,FASN,CIDEA,ACACA and SREBP1,thereby negatively regulated milk protein and fat synthesis in both normal and LPS-treated BMECs.On the contrary,UFL1 overexpression increased the level of WAP,ELF5,CSN2,FASN,CIDEA,AC AC A and SREBP1,thereby positively regulated milk protein and fat synthesis in both normal and LPS-treated BMECs.Furthermore,we found that UFL1 depletion significantly decreased the expression of mTOR phosphorylation,whereas overexpression of UFL1 increased mTOR phosphorylation.These results suggest that UFL1 could regulate milk protein and fat synthesis and cell proliferation probably via the mTOR-SREBP-1 c/Cyclin D1 signaling pathway.4.UFL1 regulates the activation of TLR4/NF-?B signaling pathway in LPS-induced bovine mammary epithelial cells.During infection,inflammatory cells are recruited to the site of damage,provoking the production of numerous cytokines by local cells expressing TLRs.NF-?B is an important transcription factor located downstream of the TLR4-mediated signaling pathway and plays an essential role in regulating immune response and cellular processes.In the present study,we first knocked down TLR4 by siRNA to confirm that the activation of NF-?B is mediated by TLR4 in LPS-stimulated BMECs,Results showed that,after transfection with si-TLR4,the expression of TLR4 and subsequent expression of NF-?B p65 in the nuclear were both muted.Furthermore,as determined by immunoblotting and immunofluorescence assay,UFL1 depletion significantly increased the expression of TLR4,phospho-NF-?B P65,and phospho-I?B?,whereas overexpression of UFL1 led to a marked reduction in the expression of TLR4,phospho-NF-?B P65,and phosphor I?B? after LPS treatment.No significant differencewas observed in the NF-?B P65 levels between the overexpression and knockdown groups under LPS treatment.These results indicate that UFL1 depletion enhances the activation of the LPS-induced TLR4/NF-?B signaling pathway,and UFL1 overexpression can suppress the activation of the TLR4/NF-?B pathway,suggesting that UFLl can control the inflammatory response and cell damage by inhibiting NF-?B activation.