- Open Access
Metabolomic analysis reveals the influence of HMBOX1 on RAW264.7 cells proliferation based on UPLC-MS/MS
BMC Genomics volume 24, Article number: 272 (2023)
Macrophages are important effector cells in tumor progression and immune regulation. Previously, we demonstrated that the transcription suppressor homeobox containing 1(HMBOX1) exhibits immunosuppressive activity in LPS-induced acute liver injury by impeding macrophage infiltration and activation. We also observed a lower proliferation in HMBOX1-overexpressed RAW264.7 cells. However, the specific mechanism was unclear. Here, a work was performed to characterize HMBOX1 function related to cell proliferation from a metabolomics standpoint by comparing the metabolic profiles of HMBOX1-overexpressed RAW264.7 cells to those of the controls. Firstly, we assessed HMBOX1 anti-proliferation activity in RAW264.7 cells with CCK8 assay and clone formation. Then, we performed metabolomic analyses by ultra-liquid chromatography coupled with mass spectrometry to explore the potential mechanisms. Our results indicated that HMBOX1 inhibited the macrophage growth curve and clone formation ability. Metabolomic analyses showed significant changes in HMBOX1-overexpressed RAW264.7 metabolites. A total of 1312 metabolites were detected, and 185 differential metabolites were identified based on the criterion of OPLS-DA VIP > 1 and p value < 0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the elevated HMBOX1 in RAW264.7 inhibited the pathways of amino acid and nucleotide metabolism. Glutamine concentrations decreased significantly in HMBOX1-overexpressed macrophages, and glutamine-related transporter SLC1A5 was also downregulated. Furthermore, SLC1A5 overexpression reversed HMBOX1 inhibition of macrophage proliferation. This study demonstrated the potential mechanism of the HMBOX1/SLC1A5 pathway in cell proliferation by regulating glutamine transportation. The results may help provide a new direction for therapeutic interventions in macrophage-related inflammatory diseases.
As important effector cells in tumor progression and immune regulation, macrophages are pivotal to tumorigenesis and development [1, 2]. Emerging evidence indicates that many macrophages are established from progenitors in the yolk sac and fetal liver, and then they are maintained with self-proliferation and monocyte supplementation [3,4,5]. Macrophage proliferation usually contributes to its accumulation in tumors or inflammatory diseases; thus, inhibiting macrophage proliferation may prevent disease progression [6, 7]. Elevated macrophage levels combined with a tumor-promoting phenotype have been linked to an undesirable outcome in patients. Unsurprisingly, macrophages have been suggested as an important target for tumor immunotherapy.
The novel human transcription suppressor homeobox containing 1 (HMBOX1) has an atypical homeobox domain containing 78 amino acids and a putative HNF1N domain , with the function in cell proliferation [9, 10], apoptosis , differentiation [12,13,14], and immunomodulation [15,16,17,18]. Previously, we found that HMBOX1 acts as a transcriptional repressor of interferon γ (IFN-γ) in natural killer (NK) cells [15, 16] and protects against LPS/D-GalN-induced acute liver injury by inhibiting liver inflammation. Further research identified negative regulation of NF-κB/CCL2 signal transduction in hepatocytes. However, the regulatory effects of HMBOX1 on macrophage activity and proliferation have not yet been studied in detail.
As the most abundant amino acid in the human body , glutamine provides an energy substrate and serves as a precursor of other amino acids, including glutamic acid, aspartate, and alanine [20,21,22,23].Other important metabolites of glutamine include ammonia, lactic acid, and pyruvic acid.
Recent reviews have highlighted the importance of glutamine function in macrophages [24,25,26], metabolically active immune cells that require glutamine and its metabolic products for protein synthesis . Moreover, glutamine metabolism profoundly influences macrophage proliferation and activation, with 10 mM glutamine reported to increase RAW264.7proliferation and viability [24, 28]. Glutamine has been shown to promote cell proliferation in general , acting through glutamine-related transporters of the solute carrier (SLC) family .
Because glutamine is hydrophilic and water-soluble , SLC transporter proteins (e.g., SLC1, SLC6, SLC7, and SLC38) help carry extracellular glutamine past the cell membrane , particularly SLC1A5 . The influence of SLC1A5 on amino acid metabolism makes it an important regulator of cell proliferation [30, 34, 35]. Other SLC1A5 substrates include aspartic acid, serine, alanine, and cysteine, all of which are also involved in cell metabolism. While we observed a lower SLC1A5 levels in HMBOX1-overexpressed RAW264.7 cells, the specific influence on macrophage biological behaviour remains unclear.
This study aimed to investigate the role of HMBOX1 in regulating macrophage proliferation by using untargeted liquid chromatography coupled with mass spectrometry (LC-MS) to profile key metabolites and characterize their variation. The profiling results allowed for clarification of the mechanism underlying HMBOX1 effects on macrophage proliferation. Our data clearly showed that HMBOX1-overexpressed macrophages display distinct metabolic signatures and associated changes in relevant metabolites. Furthermore, we have provided valuable empirical evidence of HMBOX1 function in macrophage proliferation, which is expected to be a potential therapeutic target in the treatment of tumors and inflammatory diseases.
Cell lines and cell culture
Murine macrophage cell line RAW264.7 was obtained from the Cell Bank of Type Culture Collection at the Chinese Academy of Sciences (Shanghai, China). All cells were maintained in DMEM (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Sijiqing, Hangzhou, China).
RAW264.7 cells were plated at a density of 2 × 105 cells/well in 6 plates and transfected with HMBOX1-overexpression plasmid (pcDNA3.1-HMBOX1 plasmid, YouBia, China) or blank vector for 6 h, and they were then cultured at 37 ℃ for 24 h. As for lentivirus transduction, RAW264.7 cells were plated onto six-well plates and transduced with lentivirus supernatants containing HMBOX1-overexpressed plasmid (pHBLV-CMV-MCS-fLUC-EF1-ZsGreen-T2A-PURO, Hanbio, China) for 8 h. After washing twice with phosphate-buffered saline (PBS), cells were cultured in DMEM for 48 h, after which they were cultured with 1.0 µg/mL puromycin (Sigma-Aldrich, St. Louis, MO, USA) for 5 days before use. The HMBOX1-overexpressed RAW264.7 cells were plated at density of 2 × 105 cells/well in 6 plates and transfected with SLC1A5-overexpression plasmid (pCDH3.1-CMV-MCS-EF1-Neo, Keyybio, China) or blank vector for 6 h; then, they were cultured at 37 ℃ for 24 h. Cells were then collected for the subsequent experiments.
HMBOX1-lentiviral plasmid transducted RAW264.7 were incubated with LPS (10 ng/mL) for 6 h. After washing with 3 mL PBS (Sigma-Aldrich) at 37 °C, cells were extracted (1 mL per 5 × 106 cells) using an ice-cold solution (methanol-acetonitrile-water = 40:40:20, v/v). Next, cells were treated with ultrasound for 30 min at 4 °C and centrifuged for 20 min at 14,000 × g. The supernatant was collected, vacuum-dried, redissolved with acetonitrile-water solution (1:1,v/v), and transferred into a high-performance liquid chromatography (HPLC) vial.
We performed LC-MS analysis with the Agilent 1290 Infinity UHPLC system interfaced with an AB Triple TOF 6600 (AB Sciex, Germany) and HILIC (150 × 4.6 mm, 5 μm) HPLC columns (Hi Chrom; Reading, UK). The HILIC mobile phase consisted of 25 mM ammonium acetate and 20 ammonia water in (A) HPLC-grade water and (B) acetonitrile. The solvent gradient was 95% B (0–0.5 min), 95–65% B (0.5–7 min), 65–40% B (7–8 min), and 40–95% B (9.1–12 min); it was then maintained at 95%, and the flow rate was 0.5 mL/min. Nitrogen sheath and auxiliary gas flow rates were maintained at 30 and 60 arbitrary units, respectively. The electrospray ionization interface was set to the positive/negative dual-polarity mode with a spray voltage of 5.5 kV and ion transfer capillary temperature of600°C. Full-scan data were obtained under a mass-to‐charge ratio (m/z) between 25 and 1000 amu for both ionization modes. To monitor stability and repeatability, quality control (QC) samples were prepared by pooling 10 µL of each sample. The QC samples were inserted after every five regular samples.
Data extraction and processing
Data were extracted in XCMS Online. Isotopes and adducts were annotated with Collection of Algorithms of Metabolite pRofile Annotation (CAMERA). Peaks of the resultant metabolite lists were then manually evaluated. Suitable metabolites were matched with retention times of authentic standard mixtures run in the same sequences. Metabolite identification was validated with library searches against accurate metabolite-mass data from the Human Metabolome Database and Kyoto Encyclopedia of Genes and Genomes (KEGG) [36, 37].
Data were subjected to multivariate Pareto-scaled principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) using the ropls R package. Model robustness was evaluated with seven-fold cross-validation and response permutation testing. Variable importance in the projection (VIP) for each component of the OPLS-DA model was calculated to determine its contribution to classification. Independent Student’s t-tests were used to determine between-group differences. Metabolites were considered significantly changed if VIP > 1 and p < 0.05. Relationships between variables were assessed with Pearson’s correlation analyses.
HMBOX1 effects on RAW264.7 proliferation
RAW264.7 cells were transduced with HMBOX1-overexpressed lentivirus plasmid, increased HMBOX1 level was observed (Fig. S1a). The cells were collected for CCK8 assays at 24, 48, 72, and 96 h, and the results revealed decreased proliferation in RAW264.7 cells (Fig. 1a). A clone formation assay yielded similar outcomes (Fig. 1b). These results suggest that HMBOX1 has a significant effect on RAW264.7 cell proliferation.
HMBOX1 effects on LPS-induced RAW264.7 activation
Transfected with pcDNA3.1-HMBOX1 plasmid (Fig. S1b) or the controls, RAW264.7 cells were stimulated with LPS (10 ng/mL) for 24 h. There were significant decreases in the levels of TNF-α and IL-6 in LPS-stimulated RAW264.7 cell supernatant compared to those in the control group (Fig. 2a). Meanwhile, impaired phagocytosis ability was observed in the HMBOX1-overexpressed RAW264.7 cells compared to those in the controls, with a lower percentage of neutral-red-positive macrophages (Fig. 2b). These results indicate that HMBOX1 has a significant effect on RAW264.7 cell activation.
Differential metabolites profiles between the control and HMBOX1-overexpressed groups
As shown in Fig. 3a, the total ion chromatograph (TIC) of cell metabolites in the control and HMBOX1-overexpressed groups were identified to bein the HILIC positive or negative ion mode. On this basis, we initially used Compound Discoverer software to process data and obtain a matrix which included 1312 metabolites, including lipids, nucleotides, organic acids, and organic nitrogen. The data obtained were exported into R package for multivariate statistical analysis using the PLS-DA, PCA, and OPLS-DA models. The OPLS-DA score scatterplot showed a clear distinction between the two groups (Fig. 3b). The confidence test result of the OPLS-DA model also indicated that there was no overfitting phenomenon in the OPLS-DA model (Fig. 3c).
Identification of differential metabolites in the control and HMBOX1-overexpressed groups
As shown in Fig. 4a, the volcano plot indicated that HMBOX1-overexpressed macrophages had significantly different metabolite expression (fold change [FC] > 1.5 or < 0.67 and p < 0.05). The results of KEGG analysis on differential metabolites revealed that they participated in the same metabolic processes or cellular pathways (heatmap, Fig. 4b). Additionally, HMBOX1 overexpression affected multiple types of amino acid and nucleotide metabolism (Fig. 4c), such as alanine, aspartate and glutamate metabolism, arginine and proline metabolism, glycine, serine and threonine metabolism, valine, leucine and isoleucine biosynthesis, and pyrimidine metabolism. The differential metabolites in amino acid and nucleotide metabolism are listed in Table 1. These results collectively suggest the regulatory function of HMBOX1 in RAW264.7 cell metabolism.
HMBOX1 downregulated glutamine level in RAW264.7 cells by inhibiting SLC1A5-mediated intracellular transportation
As shown in Table 1, several amino acid metabolites were significantly decreased following HMBOX1 overexpression, these metabolites have important roles in cell proliferation and biological function. Glutamine, for instance, serves as a precursor of other amino acids and is a key amino acid for macrophage proliferation and activation. HMBOX1-overexpressed RAW264.7 cells had lower glutamine levels than the control cells (Fig. 5a). Additionally, glutamine-related transporters, including SLC1A5, SLC38A1, SLC38A2, and SLC38A10, were observed to be negatively regulated by the overexpressed HMBOX1, using the LC-MS/MS method (Fig. 5b). Thus, the mechanism of HMBOX1 inhibition of macrophage proliferation appears to involve the downregulation of glutamine transporters and the limiting of glutamine intracellular transport.
Previous research reported that cancer cells took up glutamine through Slc family members, including SLC1, 6, 7, and 38 . Among them, SLC1A5 has been deeply studied and acted as an obligatory sodium-dependent transporter for neutral amino acids . SLC1A5 inhibition impaired glutamine uptake and cell multiplication capacity . To confirm SLC1A5 participation in the regulatory function of HMBOX1 in cell proliferation, we overexpressed SLC1A5 in RAW264.7 cells transducted with HMBOX1 lentiviral plasmid (Fig. S1c). A CCK8 proliferation experiment identified that SLC1A5 overexpression significantly reversed HMBOX1 inhibition on cell proliferation (Fig. 5c). Our BrdU proliferation experiment confirmed these findings, along with the restored proliferation ability of HMBOX1-overexpressed RAW264.7 cells (Fig. 5d). These results suggest that SLC1A5-mediated glutamine transport plays a major role in RAW264.7 cell proliferation and that HMBOX1/SLC1A5 is a potential target for regulating macrophage proliferation.
Although it is a fairly novel field, metabolomics has already made important contributions to research on macrophage function [41,42,43]. Here, we applied metabolomics to demonstrate that transcription repressor HMBOX1 downregulated intracellular glutamine levels to block macrophage proliferation. Proteomics and in vitro experiments indicated that the mechanism involved HMBOX1 inhibiting the expression of SLC glutamine-related transporters. These results show that HMBOX1 has an important regulatory role in macrophage-related immune diseases.
Our metabolomics analysis also revealed many other amino acids that were significantly inhibited by HMBOX1, including valine, leucine, isoleucine, glycine, serine, threonine, arginine, proline, and pyrimidine. These effects are likely attributable to the importance of glutamine metabolism as a source of material for synthesizing other intracellular components. For instance, amide nitrogen in glutamine is necessary for biosynthesis, and cells in vivo use glutamine to produce purines, pyrimidines, and other amino acids [22, 23, 44, 45]. To explore the mechanism of glutamine effects on macrophage proliferation, we cultured HMBOX1-overexpressed RAW264.7 cells in DMEM culture medium supplemented with 2mmol/L L-glutamine. However, the inhibition function of HMBOX1 on cell proliferation was not improved by the increased L-glutamine in medium (Fig. S2), suggesting that the level of glutamine in the extracellular medium was sufficient for cell growth, and the inhibition of HMBOX1 on the proliferation of RAW264.7 cells was mainly related to its suppression of the intracellular transport of glutamine, but not the level of extracellular glutamine possibly.
In our study, overexpressed HMBOX1 down-regulated the level of macrophage glutamine transporter SLC1A5. We also observed decreased glutamine level in macrophages, as well as the down-regulate metabolism of various amino acids and pyrimidine nucleotides, which was consistent with the previous reports. All of the data suggested the mechanism of HMBOX1 in inhibiting cell multiplication capacity by targeting SLC1A5-related glutamine transportation. Further study was needed to disclose the specific regulatory mechanism. Additionally, our proteomics analyses showed that HMBOX1 suppressed the expression of glutamine transporter and glutaminase (with 10% suppression) but not that of glutamine synthetase or glutamine dehydrogenase. Therefore, HMBOX1 seems to be important for glutamine transportation but not for its metabolism.
Except for the SLC family transporters, the phytosphingosine level was significantly promoted following HMBOX1 overexpression. It has been reported that phytosphingosine induces cell apoptosis via a mitochondrially mediated pathway ,which maybe another mechanism for HMBOX1function in inhibiting RAW264.7 proliferation ability. It may also be a reason for the partial recovery of cell proliferation after SLC1A5 was elevated.
In addition to promoting cell proliferation, glutamine supports macrophage immune function, including phagocytosis, pro-inflammatory cytokine synthesis/secretion, and antigen presentation [47, 48]. Jiang et al. reported that glutamine serves as a carbon and nitrogen source for the metabolic reprogramming to M1-like macrophages . Our study demonstrated that HMBOX1 significantly inhibits LPS-induced M1 activation of macrophages, with decreased M1-related cytokines and cell phagocytic ability. Evidence from this study indicates that HMBOX1 suppression of glutamine intracellular transportation is an important contributor. Therefore, HMBOX1/SLC1A5-mediated downregulation of glutamine uptake may be one mechanism for the protective effects of HMBOX1 in liver inflammation.
Elevated HMBOX1 has also been shown to influence sphingomyelin metabolism in macrophages;the lipid sphingomyelin accounts for approximately 25% of macrophage membranes , participating in phagocytosis, lysosome stabilization, receptor-mediated chemotaxis, antigen presentation [51,52,53], and regulation of TLR4-mediated innate immune responses [54, 55]. Thus, sphingomyelin metabolism may be another pathway that HMBOX1 acts upon to regulate macrophage immune function. However, the mechanism is not yet clear, and further investigation is required to confirm this hypothesis.
Our results demonstrate that HMBOX1 inhibits the proliferation and M1 activation of RAW264.7 cells by modulating the levels of many metabolites and corresponding metabolic pathways in RAW264.7 cells. Downregulated glutamine, mediated by HMBOX1-inhibited SLC1A5 function affected RAW264.7 cell proliferation and biological function. These data support our previous work on HMBOX1 inhibition function on macrophage proliferation and M1 activation. The results obtained in the present study provide the foundation for further exploration of HMBOX1 function in macrophage biological function.
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
Gunassekaran GR, Poongkavithai Vadevoo SM, Baek MC, Lee B. M1 macrophage exosomes engineered to foster M1 polarization and target the IL-4 receptor inhibit tumor growth by reprogramming tumor-associated macrophages into M1-like macrophages. Biomaterials. 2021;278:121137.
Na YR, Je S, Seok SH. Metabolic features of macrophages in inflammatory diseases and cancer. Cancer Lett. 2018;413:46–58.
Ito C, Hikosaka-Kuniishi M, Yamazaki H, Yamane T. Multiple cell populations generate macrophage progenitors in the early yolk sac. Cell Mol Life Sci. 2022;79(3):159.
Lakhdari O, Yamamura A, Hernandez GE, Anderson KK, Lund SJ, Oppong-Nonterah GO, Hoffman HM, Prince LS. Differential Immune activation in fetal macrophage populations. Sci Rep. 2019;9(1):7677.
van de Laar L, Saelens W, De Prijck S, Martens L, Scott CL, Van Isterdael G, Hoffmann E, Beyaert R, Saeys Y, Lambrecht BN, et al. Yolk sac macrophages, fetal liver, and adult monocytes can colonize an empty niche and develop into functional Tissue-Resident Macrophages. Immunity. 2016;44(4):755–68.
Wang J, Wang Y, Chu Y, Li Z, Yu X, Huang Z, Xu J, Zheng L. Tumor-derived adenosine promotes macrophage proliferation in human hepatocellular carcinoma. J Hepatol. 2021;74(3):627–37.
Tang J, Frey JM, Wilson CL, Moncada-Pazos A, Levet C, Freeman M, Rosenfeld ME, Stanley ER, Raines EW, Bornfeldt KE. Neutrophil and Macrophage Cell Surface Colony-Stimulating Factor 1 Shed by ADAM17 Drives Mouse Macrophage Proliferation in Acute and Chronic Inflammation. Mol Cell Biol 2018, 38(17).
Chen S, Saiyin H, Zeng X, Xi J, Liu X, Li X, Yu L. Isolation and functional analysis of human HMBOX1, a homeobox containing protein with transcriptional repressor activity. Cytogenet Genome Res. 2006;114(2):131–6.
Yu YL, Diao NN, Li YZ, Meng XH, Jiao WL, Feng JB, Liu ZP, Lu N. Low expression level of HMBOX1 in high-grade serous ovarian cancer accelerates cell proliferation by inhibiting cell apoptosis. Biochem Biophys Res Commun. 2018;501(2):380–6.
Zhou J, Wang M, Deng D. c-Fos/microRNA-18a feedback loop modulates the tumor growth via HMBOX1 in human gliomas. Biomed Pharmacother. 2018;107:1705–11.
Ma H, Su L, He X, Miao J. Loss of HMBOX1 promotes LPS-induced apoptosis and inhibits LPS-induced autophagy of vascular endothelial cells in mouse. Apoptosis: an international journal on programmed cell death. 2019;24(11–12):946–57.
Han L, Shao J, Su L, Gao J, Wang S, Zhang Y, Zhang S, Zhao B, Miao J. A chemical small molecule induces mouse embryonic stem cell differentiation into functional vascular endothelial cells via Hmbox1. Stem Cells Dev. 2012;21(15):2762–9.
Lu W, Su L, Yu Z, Zhang S, Miao J. The New Role of CD163 in the Differentiation of Bone Marrow Stromal Cells into Vascular Endothelial-Like Cells. Stem Cells Int 2016, 2016:2539781.
Ma H, Su L, Zhang S, Kung H, Miao J. Inhibition of ANXA7 GTPase activity by a small molecule promotes HMBOX1 translation of vascular endothelial cells in vitro and in vivo. Int J Biochem Cell Biol. 2016;79:33–40.
Wu L, Zhang C, Zhang J. HMBOX1 negatively regulates NK cell functions by suppressing the NKG2D/DAP10 signaling pathway. Cell Mol Immunol. 2011;8(5):433–40.
Wu L, Zhang C, Zheng X, Tian Z, Zhang J. HMBOX1, homeobox transcription factor, negatively regulates interferon-γ production in natural killer cells. Int Immunopharmacol. 2011;11(11):1895–900.
Zhao H, Han Q, Lu N, Xu D, Tian Z, Zhang J. HMBOX1 in hepatocytes attenuates LPS/D-GalN-induced liver injury by inhibiting macrophage infiltration and activation. Mol Immunol. 2018;101:303–11.
Gong J, Liu R, Zhuang R, Zhang Y, Fang L, Xu Z, Jin L, Wang T, Song C, Yang K, et al. miR-30c-1* promotes natural killer cell cytotoxicity against human hepatoma cells by targeting the transcription factor HMBOX1. Cancer Sci. 2012;103(4):645–52.
Rogero MM, Borelli P, Fock RA, de Oliveira Pires IS, Tirapegui J. Glutamine in vitro supplementation partly reverses impaired macrophage function resulting from early weaning in mice. Nutrition. 2008;24(6):589–98.
Grohmann U, Mondanelli G, Belladonna ML, Orabona C, Pallotta MT, Iacono A, Puccetti P, Volpi C. Amino-acid sensing and degrading pathways in immune regulation. Cytokine Growth Factor Rev. 2017;35:37–45.
Cruzat V, Macedo Rogero M, Noel Keane K, Curi R, Newsholme P. Glutamine: Metabolism and Immune Function, Supplementation and Clinical Translation. Nutrients 2018, 10(11).
Curi R, Newsholme P, Marzuca-Nassr GN, Takahashi HK, Hirabara SM, Cruzat V, Krause M, de Bittencourt PI. Jr.: Regulatory principles in metabolism-then and now. Biochem J 2016, 473(13):1845–1857.
Cruzat VF, Pantaleão LC, Donato J Jr, de Bittencourt PI Jr, Tirapegui J. Oral supplementations with free and dipeptide forms of L-glutamine in endotoxemic mice: effects on muscle glutamine-glutathione axis and heat shock proteins. J Nutr Biochem. 2014;25(3):345–52.
Sartori T, Galvão Dos Santos G, Nogueira-Pedro A, Makiyama E, Rogero MM, Borelli P, Fock RA. Effects of glutamine, taurine and their association on inflammatory pathway markers in macrophages. Inflammopharmacology. 2018;26(3):829–38.
Ren W, Xia Y, Chen S, Wu G, Bazer FW, Zhou B, Tan B, Zhu G, Deng J, Yin Y. Glutamine metabolism in Macrophages: a novel target for Obesity/Type 2 diabetes. Adv Nutr. 2019;10(2):321–30.
Tan HWS, Sim AYL, Long YC. Glutamine metabolism regulates autophagy-dependent mTORC1 reactivation during amino acid starvation. Nat Commun. 2017;8(1):338.
Nagy C, Haschemi A. Time and demand are two critical dimensions of Immunometabolism: the process of macrophage activation and the Pentose phosphate pathway. Front Immunol. 2015;6:164.
Nelson VL, Nguyen HCB, Garcìa-Cañaveras JC, Briggs ER, Ho WY, DiSpirito JR, Marinis JM, Hill DA, Lazar MA. PPARγ is a nexus controlling alternative activation of macrophages via glutamine metabolism. Genes Dev. 2018;32(15–16):1035–44.
Kim B, Li J, Jang C, Arany Z. Glutamine fuels proliferation but not migration of endothelial cells. Embo j. 2017;36(16):2321–33.
Osman I, He X, Liu J, Dong K, Wen T, Zhang F, Yu L, Hu G, Xin H, Zhang W, et al. TEAD1 (TEA domain transcription factor 1) promotes smooth muscle cell proliferation through upregulating SLC1A5 (solute Carrier Family 1 Member 5)-Mediated glutamine uptake. Circul Res. 2019;124(9):1309–22.
Dong J, Xiao D, Zhao Z, Ren P, Li C, Hu Y, Shi J, Su H, Wang L, Liu H, et al. Epigenetic silencing of microRNA-137 enhances ASCT2 expression and tumor glutamine metabolism. Oncogenesis. 2017;6(7):e356.
Bhutia YD, Ganapathy V. Glutamine transporters in mammalian cells and their functions in physiology and cancer. Biochim Biophys Acta. 2016;1863(10):2531–9.
Cormerais Y, Massard PA, Vucetic M, Giuliano S, Tambutté E, Durivault J, Vial V, Endou H, Wempe MF, Parks SK, et al. The glutamine transporter ASCT2 (SLC1A5) promotes tumor growth independently of the amino acid transporter LAT1 (SLC7A5). J Biol Chem. 2018;293(8):2877–87.
Suzuki M, Toki H, Furuya A, Ando H. Establishment of monoclonal antibodies against cell surface domains of ASCT2/SLC1A5 and their inhibition of glutamine-dependent tumor cell growth. Biochem Biophys Res Commun. 2017;482(4):651–7.
Scalise M, Pochini L, Console L, Losso MA, Indiveri C. The human SLC1A5 (ASCT2) amino acid transporter: from function to structure and role in Cell Biology. Front Cell Dev Biol. 2018;6:96.
Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27–30.
Kanehisa M. Toward understanding the origin and evolution of cellular organisms. Protein Sci. 2019;28(11):1947–51.
Pochini L, Scalise M, Galluccio M, Indiveri C. Membrane transporters for the special amino acid glutamine: structure/function relationships and relevance to human health. Front Chem. 2014;2:61.
Yoo HC, Park SJ, Nam M, Kang J, Kim K, Yeo JH, Kim JK, Heo Y, Lee HS, Lee MY, et al. A variant of SLC1A5 is a mitochondrial glutamine transporter for metabolic reprogramming in Cancer cells. Cell Metab. 2020;31(2):267–283e212.
Hassanein M, Hoeksema MD, Shiota M, Qian J, Harris BK, Chen H, Clark JE, Alborn WE, Eisenberg R, Massion PP. SLC1A5 mediates glutamine transport required for lung cancer cell growth and survival. Clin Cancer Res. 2013;19(3):560–70.
Cambeiro-Pérez N, González-Gómez X, González-Barreiro C, Pérez-Gregorio MR, Fernandes I, Mateus N, de Freitas V, Sánchez B, Martínez-Carballo E. Metabolomics Insights of the Immunomodulatory Activities of Phlorizin and Phloretin on Human THP-1 Macrophages. Molecules 2021, 26(4).
Cambeiro-Pérez N, Figueiredo-González M, Pérez-Gregorio MR, Bessa-Pereira C, De Freitas V, Sánchez B, Martínez-Carballo E. Unravelling the immunomodulatory role of apple phenolic rich extracts on human THP-1- derived macrophages using multiplatform metabolomics. Food Res Int. 2022;155:111037.
Abuawad A, Mbadugha C, Ghaemmaghami AM, Kim DH. Metabolic characterisation of THP-1 macrophage polarisation using LC-MS-based metabolite profiling. Metabolomics. 2020;16(3):33.
Metzler B, Gfeller P, Guinet E. Restricting glutamine or glutamine-dependent purine and pyrimidine syntheses promotes human T cells with high FOXP3 expression and Regulatory Properties. J Immunol. 2016;196(9):3618–30.
Zhu Y, Li T, Ramos da Silva S, Lee JJ, Lu C, Eoh H, Jung JU, Gao SJ. A Critical Role of Glutamine and Asparagine γ-Nitrogen in Nucleotide Biosynthesis in Cancer Cells Hijacked by an Oncogenic Virus. mBio 2017, 8(4).
Li J, Wen J, Sun C, Zhou Y, Xu J, MacIsaac HJ, Chang X, Cui Q. Phytosphingosine-induced cell apoptosis via a mitochondrially mediated pathway. Toxicology. 2022;482:153370.
Jha AK, Huang SC, Sergushichev A, Lampropoulou V, Ivanova Y, Loginicheva E, Chmielewski K, Stewart KM, Ashall J, Everts B, et al. Network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization. Immunity. 2015;42(3):419–30.
Davies LC, Rice CM, Palmieri EM, Taylor PR, Kuhns DB, McVicar DW. Peritoneal tissue-resident macrophages are metabolically poised to engage microbes using tissue-niche fuels. Nat Commun. 2017;8(1):2074.
Jiang Q, Qiu Y, Kurland IJ, Drlica K, Subbian S, Tyagi S, Shi L. Glutamine is required for M1-like polarization of Macrophages in response to Mycobacterium tuberculosis infection. mBio. 2022;13(4):e0127422.
Sims K, Haynes CA, Kelly S, Allegood JC, Wang E, Momin A, Leipelt M, Reichart D, Glass CK, Sullards MC, et al. Kdo2-lipid A, a TLR4-specific agonist, induces de novo sphingolipid biosynthesis in RAW264.7 macrophages, which is essential for induction of autophagy. J Biol Chem. 2010;285(49):38568–79.
Denard B, Han S, Kim J, Ross EM, Ye J. Regulating G protein-coupled receptors by topological inversion. Elife 2019, 8.
Jongsma MLM, de Waard AA, Raaben M, Zhang T, Cabukusta B, Platzer R, Blomen VA, Xagara A, Verkerk T, Bliss S, et al. The SPPL3-Defined glycosphingolipid repertoire orchestrates HLA class I-Mediated Immune responses. Immunity. 2021;54(1):132–150e139.
Niekamp P, Guzman G, Leier HC, Rashidfarrokhi A, Richina V, Pott F, Barisch C, Holthuis JCM, Tafesse FG. Sphingomyelin Biosynthesis Is Essential for Phagocytic Signaling during Mycobacterium tuberculosis Host Cell Entry. mBio 2021, 12(1).
Olona A, Hateley C, Muralidharan S, Wenk MR, Torta F, Behmoaras J. Sphingolipid metabolism during toll-like receptor 4 (TLR4)-mediated macrophage activation. Br J Pharmacol. 2021;178(23):4575–87.
Köberlin MS, Snijder B, Heinz LX, Baumann CL, Fauster A, Vladimer GI, Gavin AC, Superti-Furga G. A conserved Circular Network of Coregulated lipids modulates Innate Immune responses. Cell. 2015;162(1):170–83.
We would like to thank Applied Protein Technology Company for technical support. We would like to thank Editage (www.editage.cn) for English language editing.
This research was funded by the Natural Science Foundation of China (81901610) and the Jinan Technology Development Program (202019118).
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Jiang, W., Jiang, Y., Zhang, X. et al. Metabolomic analysis reveals the influence of HMBOX1 on RAW264.7 cells proliferation based on UPLC-MS/MS. BMC Genomics 24, 272 (2023). https://doi.org/10.1186/s12864-023-09361-x