LAP3 contributes to IFN-γ-induced arginine depletion and malignant transformation of bovine mammary epithelial cells

Abstract

Background IFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown. Methods In this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry. Results The targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control. Conclusions These results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.

Metabolomics and Lipidomics with Mass Spectrometry Imaging Reveal Mechanistic Insights into Dibutyl Phthalate-Promoted Proliferation of Breast Cancer Cell Spheroids

“…Glutamine is the most consumed amino acid, which fuels the tricarboxylic acid cycle, nucleotide synthesis, and lipid metabolism in breast cancer cells. , Glutamate coupled with cysteine is utilized to synthesize glutathione which promotes cellular survival and proliferation and resists apoptosis through bolstering cell antioxidant defense and maintaining redox homeostasis. − Methionine, a precursor for glutathione, can metabolize to S-adenosylmethionine which provides methyl donors for creatine production, one of the end products for arginine catabolism. , Creatine can be reversibly phosphorylated to phosphocreatine to enhance the energy pool. The significantly decreased arginine and citrulline levels and increased levels of downstream metabolites such as creatine, glutamine, and glutamate indicated that DBP exposure may stimulate arginine catabolism, which is consistent with the epidemiological studies showing significantly lower arginine levels in breast tumor tissues than in adjacent normal tissues. , Therefore, the upregulation of glutamine, methionine, glutathione, and creatine indicated the increase of energy supply and storage and the enhancement of antioxidant defense system in breast CCS after DBP exposure.…”

“…The significantly decreased arginine and citrulline levels and increased levels of downstream metabolites such as creatine, glutamine, and glutamate indicated that DBP exposure may stimulate arginine catabolism, which is consistent with the epidemiological studies showing significantly lower arginine levels in breast tumor tissues than in adjacent normal tissues. 43,44 Therefore, the upregulation of glutamine, methionine, glutathione, and creatine indicated the increase of energy supply and storage and the enhancement of antioxidant defense system in breast CCS after DBP exposure. DBP-Induced Increase of GP metabolism.…”

Untargeted metabolomics reveals alternations in metabolism of bovine mammary epithelial cells upon IFN-γ treatment

“…Previously we have shown that IFN-γ treatment did not affect ornithine level, but led to reduced intracellular levels of arginine and citrulline (Fig. S 7 ), two of which are the key metabolites for arginine metabolism [ 25 ]. As a vital intermediate product for arginine synthesis, we further detected the intracellular level of argininosuccinate in this study.…”

“…In BMECs, IFN-γ accelerates cell growth and induces malignant transformation through arginine depletion [ 16 , 28 ]. It’s been shown that IFN-γ could disturb arginine metabolism by affecting the expression of key time-limiting enzymes ASS1 [ 25 , 32 ]. In this study, the content of spermine was downregulated, which further confirms these results and suggest that the IFN-γ-induced malignant transformation of BMECs might possibly be associated with increased spermine levels in cells.…”

“…Sample preparation for targeted metabolomics was performed as previously reported [ 25 ]. Reagents and materials for targeted metabolomics analysis are the same as those for nontargeted metabolomics.…”