The Mechanism Of IFN-γ-derived Arginine Depletion Mediated By LAP3 In Bovine Mammary Epithelial Cells

Background and Objective: The etiology of breast cancer is complex,which seriously threatens the life and health safety of women in China.To clarify the mechanism of malignant transformation of healthy mammary epithelial cells is very important for the prevention and treatment of breast cancer.IFN-γ is a multifunctional cytokine.In recent years,studies have reported that the continuous increase of IFN-γ in the body is affected by diet preference and chronic inflammation,which may be one of the inducers of cancer.The previous research found that long-term lowlevel IFN-γ stimulation can induce arginine depletion and malignant transformation in bovine mammary epithelial cells(BMECs).Supplementation of arginine can adersely affect IFN-γ of BMECs malignant induction,the results has also been verified in a mice model.Therefore,it is necessary to explore the internal mechanism of arginine depletion caused by IFN-γ.Combining the transcriptomics data of BMECs in the IFN-γ treatment group and the control group obtained from the previous work,we further found that leucine aminopeptidase 3(LAP3)is a key molecule that mediates the depletion of arginine induced by IFN-γ.This project aims to reveal the molecular mechanism of the regulation of arginine depletion by IFN-γ through LAP3 from the metabolic perspective,so as to provide a new cognitive theory for the occurrence of breast cancer,and to provide help for its prevention,early diagnosis and treatment options selection.Methods: The expression and enzyme activity of LAP3 and arginine level in bovine mammary epithelial cells(MAC-T)were detected by q PCR,Western blot,ELISA and high performance liquid chromatography mass spectrometry.Nontargeted metabolomics detects changes in intracellular metabolites under different IFN-γ action times(6 h,12 h,and 24 h),and screens differential metabolites.The effect of IFN-γ treatment on intracellular arginine metabolism with or without LAP3 inhibitors were detected by targeted metabolomics.MACT cells were pretreated with IFN-γ signaling pathway inhibitors and LAP3 inhibitors.The signaling pathway that IFN-γ upregulates LAP3 and the molecular mechanism of LAP3-mediated IFN-γ regulation of arginine metabolism were screened and verified by q PCR,Western blot,and ELISA.Results:(1)The transcription and translation levels of LAP3 increase under the induction of IFN-γ,but the enzyme activity of LAP3 remains unchanged.Inhibiting the expression of LAP3 could antagonize the depletion of arginine induced by IFN-γ.(2)The results of non-targeted metabolomics testing showed that different IFN-γ treatment times can cause significant changes in intracellular metabolism,among which lipid metabolism changes the most.(3)Further verification by targeted metabolomics: the citrulline level in BMECs cells decreased under the action of IFN-γ,and the level of ornithine was not statistically different.The arginine catabolites putrescine,agmatine,spermine,creatine,glutamate and glutamine were significantly increased.The use of LAP3 inhibitors increased the intracellular citrulline level under IFN-γ treatment,but did not affect the levels of other arginine breakdown products.(4)IFN-γ activated the p38 signal pathway,blocking the pathway could down-regulate the expression of LAP3.The activation of the ERK pathway was inhibited by IFN-γ,which can up-regulate the protein expression of LAP3.Compared with the single ERK inhibitor treatment group,the co-treatment of ERK inhibitor and IFN-γ could further increase the expression of LAP3.(5)IFN-γ down-regulated the protein expression of the key enzymes of arginine anabolism,Ornithine transcarbamoylase(OTC)and Argininosuccinatesynthase 1(ASS1),and inhibition of LAP3 can antagonize the inhibitory effect of IFN-γ on OTC and ASS1.Conclusion:IFN-γ increases the expression of LAP3 in MAC-T cells by activating the p38 MAPK signaling pathway and inhibiting the ERK MAPK signaling pathway.LAP3 inhibites the expression of both OTC and ASS1,reduces citrulline production and further inhibits the conversion of citrulline to arginine,leading to inhibition of intracellular arginine anabolism.In addition,IFN-γ can also promote the catabolism of arginine in cells.The resule is decreased arginine synthesis and increased arginine decomposition in cells,resulting in arginine depletion.This study reveals the molecular mechanism of IFN-γ-induced arginine depletion in mammary epithelial cells from the perspective of metabolism,and provides a new research basis for chronic inflammation-induced cancer,which may be helpful for the diagnosis and treatment of breast cancer.