Mechanism Of IFN-γ-induced Arginine Depletion Mediated By CARS2 In Bovine Mammary Epithelial Cells

The ability of milk secretion and milk quality of dairy cows are the key factors to evaluate the economic value of dairy cows.However,the quality of milk can be easily affected by various conditions.Our previous research reported that corn stalk feeding would increase the expression of inflammatory cytokine IFN-γ and its receptor in the mammary tissue of Holstein dairy cows,inducing arginine depletion and causing autophagy in bovine mammary epithelial cells(BMECs),thereby finally deteriorating milk quality and aggravating bovine mastitis.However,the mechanism by which IFN-γ induced arginine depletion is poorly understood.Preliminary transcriptomic data suggested that mitochondrial cysteinyl-tRNA synthetase 2(CARS2)may be a key protein that mediates IFN-γ-induced arginine metabolism.According to the transcriptomic data of BMECs exposed to IFN-γ for 0,5 and 20 generations,we found that Cysteinyl-tRNA synthetase 2(CARS2),as a target protein in amino acid metabolism,may mediate the regulation of arginine metabolism by IFN-γ.Therefore,this topic focuses on the function and mechanism of CARS2 in IFN-γ-induced arginine depletion in MAC-T,thus providing a certain theoretical basis on nutritional metabolism interference,milk quality improvement,and various diseases prevention and treatment caused by arginine deficiency.In order to clarify that CARS2 is involved in IFN-γ-induced arginine depletion,q PCR and Western blotting were used to detect the expression of CARS2 after IFN-γtreatment in MAC-T;ELISA was used to detect the enzymatic activity of CARS2 and the content of intracellular arginine;Cellular amino acid metabolism was detected by HPLC-MS.The results showed that after the treatment of 10 ng/m L IFN-γ,both m RNA and protein expression of CARS2 in primary BMECs and MAC-T decreased,but the enzymatic activity did not change significantly.Without IFN-γ stimulation,arginine concentration decreased markedly after solely CARS2 knockdown.Concentration of intracellular arginine and citrulline decreased,while agmatine increased after adding IFN-γ and knocking down CARS2.In order to understand the regulation of CARS2 on the key enzymes of arginine metabolism,Western blotting and ELISA were used to detect the expression and enzymatic activity of arginine synthesis-Argininosuccinate synthase 1(ASS1),Ornithine transcarbamoylase(OTC),arginine catabolism key enzyme-Arginine decarboxylation(ADC).The results showed that IFN-γ addition and CARS2knock-down could both down-regulate the expression of ASS1 and OTC in primary BMECs and MAC-T cells,while up-regulate the expression of ADC.Both IFN-γaddition and CARS2 knock-down up-regulated the enzymatic activity of ADC in MAC-T cells and down-regulated the enzymatic activity of OTC,thereby accelerating the catabolism of arginine and inhibiting the synthesis of arginine.In order to reveal the signaling pathway that CARS2 inhibits the synthesis of ASS1 in MAC-T cells under the effect of IFN-γ,small molecule signaling pathway inhibitors were used in vitro,and the changes of p38,ERK and JNK MAPK signaling pathways were measured by real-time quantitative PCR and western blotting.It was found that IFN-γ can activate p38 and JNK molecules and inhibit the expression of ERK,next the activation of p38 and JNK molecules or the inhibition of ERK can further inhibit the expression of CARS2 and ASS1 at the transcriptional and translational levels.p38/JNK and ERK MAPK regulate its downstream molecules-CARS2 and ASS1,which eventually lead to the lack of ASS1 and the inability to synthesize sufficient arginine.In order to explore the effect of CARS2 on the proliferation,cell cycle and autophagy of BMECs,experiments such as CCK-8 activity detection and plate cloning assay were performed.The results showed that inhibition of CARS2 could not significantly change the growth activity of MAC-T,but promote the clonal proliferation of malignantly BMECs.After knockdown of CARS2,the expression of Cyclin A1 and D1 increased in malignantly proliferating BMECs.Combined with q PCR and immunofluorescence,it was found that the protein expression of LC3A/B and ATG5 were markedly increased after CARS2 interference,and the expression of p62 and Beclin-1 were significantly down-regulated,indicating that inhibition of CARS2 may promote cell proliferation and induce autophagy.To sum up,this study confirmed that CARS2 mediates IFN-γ-induced arginine depletion in bovine mammary epithelial cells,and the mechanism is that IFN-γinhibited the expression of CARS2 through p38/JNK/ERK MAPK signaling pathway and down-regulated OTC-citrulline-ASS1 pathway,inhibited the synthesis of arginine,and up-regulated ADC-agmatine to promote the catabolism of arginine.This study provides the basis and ideas for the prevention and treatment of arginine metabolism-related diseases caused by inflammatory factors.