Glutamine promotes the proliferation of intestinal stem cells via inhibition of TP53-induced glycolysis and apoptosis regulator promoter methylation in burned mice

Abstract

Background: Intestinal stem cells (ISCs) play a pivotal role in maintaining intestinal homeostasis and facilitating the restoration of intestinal mucosal barrier integrity. Glutamine (Gln) is a crucial energy substrate in the intestine, promoting the proliferation of ISCs and mitigating damage to the intestinal mucosal barrier after burn injury. However, the underlying mechanism has not yet been fully elucidated. The objective of this study was to explore the mechanism by which Gln facilitates the proliferation of ISCs. Methods: A mouse burn model was established to investigate the impact of Gln on intestinal function. Subsequently, crypts were isolated, and changes in TP53-induced glycolysis and apoptosis regulator (TIGAR) expression were assessed using real-time quantitative polymerase chain reaction (RT-qPCR), western blotting, immunohistochemistry, and immunofluorescence. The effects of TIGAR on cell proliferation were validated through CCK-8, EdU, and clonogenicity assays. Furthermore, the effect of TIGAR on Yes-associated protein (YAP) nuclear translocation and ferroptosis was examined by western blotting and immunofluorescence staining. Finally, dot blot analysis and methylation-specific PCR were performed to evaluate the effect of Gln on TIGAR promoter methylation. Results: The mRNA and protein levels of TIGAR decreased after burn injury, and supplementation with Gln increased the expression of TIGAR. TIGAR accelerates the nuclear translocation of YAP, thereby increasing the proliferation of ISCs. Concurrently, TIGAR promotes the synthesis of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione to suppress ferroptosis in ISCs. Subsequent investigations demonstrated that Gln inhibits TIGAR promoter methylation by increasing the expression of the demethylase ten-eleven translocation. This change increased TIGAR transcription, increased NADPH synthesis, and reduced oxidative stress, thereby facilitating the restoration of intestinal mucosal barrier integrity post-burn injury. Conclusions: Our data confirmed the inhibitory effect of Gln on TIGAR promoter methylation, which facilitates YAP translocation into the nucleus and suppresses ferroptosis, ultimately promoting the proliferation of ISCs.

Keywords

Burn injury / Intestinal stem cells / Glutamine / TP53-induced glycolysis and apoptosis regulator / Methylation / Proliferation / Ferroptosis

References

[1] Knuth CM, Auger C, Jeschke MG. Burn-induced hypermetabolism and skeletal muscle dysfunction. Am J Physiol Cell Physiol 2021; 321: C58-c71. https://doi.org/10.1152/ajpcell.00106.2021.

[2] Huang J, Chen Y, Guo Z, Yu Y, Zhang Y, Li P, et al. Prospective study and validation of early warning marker discovery based on integrating multi-omics analysis in severe burn patients with sepsis. Burns Trauma 2023; 11: tkac050. https://doi.org/10.1093/burnst/tkac050.

[3] Zha X, Su S, Wu D, Zhang P, Wei Y, Fan S, et al. The impact of gut microbiota changes on the intestinal mucus barrier in burned mice: a study using 16S rRNA and metagenomic sequencing. Burns Trauma 2023; 11: tkad056. https://doi.org/10.1093/burnst/tkad056.

[4] Qin C, Jiang Y, Chen X, Bian Y, Wang Y, Xie K, et al. Dexmedetomidine protects against burn-induced intestinal barrier injury via the MLCK/p-MLC signalling pathway. Burns J Int Soc Burn Inj 2021; 47: 1576-85. https://doi.org/10.1016/j.burns.2021.01.017.

[5] Luo H, Li M, Wang F, Yang Y, Wang Q, Zhao Y, et al. The role of intestinal stem cell within gut homeostasis: focusing on its interplay with gut microbiota and the regulating pathways. Int J Biol Sci 2022; 18: 5185-206. https://doi.org/10.7150/ijbs.72600.

[6] Beckmann N, Pugh AM, Caldwell CC. Burn injury alters the intestinal microbiome’s taxonomic composition and functional gene expression. PLoS One 2018; 13: e0205307. https://doi.org/10.1371/journal.pone.0205307.

[7] Xi P, Hui TL, Yi YZ, Pei W, Liang WS. Relationship between enterogenous hypermetabolism and intestine injury after severe burn injury. Acta Acade Med Militaris Tertiae 2003; 25: 1745-7.

[8] Pentinmikko N, Katajisto P. The role of stem cell niche in intestinal aging. Mech Ageing Dev 2020; 191: 111330. https://doi.org/10.1016/j.mad.2020.111330.

[9] Sphyris N, Hodder MC, Sansom OJ. Subversion of niche-signalling pathways in colorectal cancer: what makes and breaks the intestinal stem cell. Cancers 2021; 13: 1000. https://doi.org/10.3390/cancers13051000.

[10] Nath A, Chakrabarti P, Sen S, Barui A.Reactive oxygen species in modulating intestinal stem cell dynamics and function. Stem Cell Rev Rep 2022; 18: 2328-50. https://doi.org/10.1007/s12015-022-10377-1.

[11] Wang D, Li P, Odle J, Lin X, Zhao J, Xiao K, et al. Modulation of intestinal stem cell homeostasis by nutrients: a novel therapeutic option for intestinal diseases. Nutr Res Rev 2022; 35: 150-8. https://doi.org/10.1017/S0954422421000172.

[12] Morris O, Jasper H. Reactive oxygen species in intestinal stem cell metabolism, fate and function. Free Radic Biol Med 2021; 166: 140-6. https://doi.org/10.1016/j.freeradbiomed.2021.02.015.

[13] Chen Y, Tsai YH, Tseng BJ, Tseng SH. Influence of growth hormone and glutamine on intestinal stem cells: a narrative review. Nutrients 2019; 11: 1941. https://doi.org/10.3390/nu11081941.

[14] Ma N, Chen X, Liu C, Sun Y, Johnston LJ, Ma X. Dietary nutrition regulates intestinal stem cell homeostasis. Crit Rev Food Sci Nutr 2022; 63: 11263-74. https://doi.org/10.1080/10408398.2022.2087052.

[15] Amirato GR, Borges JO, Marques DL, Santos JMB, Santos CAF, Andrade MS, et al. L-glutamine supplementation enhances strength and power of knee muscles and improves Glycemia control and plasma redox balance in exercising elderly women. Nutrients 2021; 13: 1025. https://doi.org/10.3390/nu13031025.

[16] Wischmeyer PE.Glutamine in burn injury. Nutr Clin Prac 2019; 34: 681-7. https://doi.org/10.1002/ncp.10362.

[17] Xiao Q, Chen YH, Pratama SA, Chen YL, Shirakawa H, Peng HC, et al. The prophylactic effects of glutamine on muscle protein synthesis and degradation in rats with ethanol-induced liver damage. Nutrients 2021; 13: 2788. https://doi.org/10.3390/nu13082788.

[18] DeBerardinis RJ, Cheng T. Q’s next: the diverse functions of glutamine in metabolism, cell biology and cancer. Oncogene 2010; 29: 313-24. https://doi.org/10.1038/onc.2009.358.

[19] Wu D, Su S, Zha X, Wei Y, Yang G, Huang Q, et al. Glutamine promotes O-GlcNAcylation of G6PD and inhibits AGR2 S-glutathionylation to maintain the intestinal mucus barrier in burned septic mice. Redox Biol 2023; 59: 102581. https://doi.org/10.1016/j.redox.2022.102581.

[20] Bensaad K, Tsuruta A, Selak MA, Vidal MN, Nakano K, Bartrons R, et al. TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell 2006; 126: 107-20. https://doi.org/10.1016/j.cell.2006.05.036.

[21] Paneque A, Fortus H, Zheng J, Werlen G, Jacinto E. The Hexosamine biosynthesis pathway: regulation and function. Genes 2023; 14: 933. https://doi.org/10.3390/genes14040933.

[22] Zhu Y, Chen X, Lu Y, Xia L, Fan S, Huang Q, et al. Glutamine mitigates murine burn sepsis by supporting macrophage M2 polarization through repressing the SIRT5-mediated desuccinylation of pyruvate dehydrogenase. Burns Trauma 2022; 10: tkac041. https://doi.org/10.1093/burnst/tkac041.

[23] Wu H, Chen QY, Wang WZ, Chu S, Liu XX, Liu YJ, et al. Compound sophorae decoction enhances intestinal barrier function of dextran sodium sulfate induced colitis via regulating notch signaling pathway in mice. Biomed Pharmacother 2021; 133: 110937. https://doi.org/10.1016/j.biopha.2020.110937.

[24] Casanova-Maldonado I, Arancibia D, Lois P, Peña-Villalobos I, Palma V. Hyperbaric oxygen treatment increases intestinal stem cell proliferation through the mTORC1/S6K1 signaling pathway in Mus musculus. Biol Res 2023; 56: 41. https://doi.org/10.1186/s40659-023-00444-3.

[25] Tang J, Chen L, Qin ZH, Sheng R. Structure, regulation, and biological functions of TIGAR and its role in diseases. Acta Pharmacol Sin 2021; 42: 1547-55. https://doi.org/10.1038/s41401-020-00588-y.

[26] Chen X, Zhang P, Zhang Y, Fan S, Wei Y, Yang Z, et al. Potential effect of glutamine in the improvement of intestinal stem cell proliferation and the alleviation of burn-induced intestinal injury via activating YAP: a preliminary study. Nutrients 2023; 15: 1766. https://doi.org/10.3390/nu15071766.

[27] Madan E, Gogna R, Kuppusamy P, Bhatt M, Pati U, Mahdi AA. TIGAR induces p53-mediated cell-cycle arrest by regulation of RB-E2F1 complex. Br J Cancer 2012; 107: 516-26. https://doi.org/10.1038/bjc.2012.260.

[28] Ni W, Yao S, Zhou Y, Liu Y, Huang P, Zhou A, et al. Long noncoding RNA GAS5 inhibits progression of colorectal cancer by interacting with and triggering YAP phosphorylation and degradation and is negatively regulated by the m(6)a reader YTHDF3. Mol Cancer 2019; 18: 143. https://doi.org/10.1186/s12943-019-1079-y.

[29] Han S, Lim JY, Cho K, Lee HW, Park JY, Ro SW, et al. Anticancer effects of YAP inhibitor (CA3) in combination with Sorafenib against hepatocellular carcinoma (HCC) in patient-derived multicellular tumor spheroid models (MCTS). Cancers 2022; 14: 2733. https://doi.org/10.3390/cancers14112733.

[30] Li D, Wang Y, Dong C, Chen T, Dong A, Ren J, et al. CST1 inhibits ferroptosis and promotes gastric cancer metastasis by regulating GPX4 protein stability via OTUB1. Oncogene 2023; 42: 83-98. https://doi.org/10.1038/s41388-022-02537-x.

[31] He BF, Wu YX, Hu WP, Hua JL, Han Y, Zhang J. ROS induced the Rab26 promoter hypermethylation to promote cigarette smoking-induced airway epithelial inflammation of COPD through activation of MAPK signaling. Free Radic Biol Med 2023; 195: 359-70. https://doi.org/10.1016/j.freeradbiomed.2023.01.001.

[32] Takeshima H, Niwa T, Yamashita S, Takamura-Enya T, Iida N, Wakabayashi M, et al. TET repression and increased DNMT activity synergistically induce aberrant DNA methylation. J Clin Invest 2020; 130: 5370-9. https://doi.org/10.1172/JCI124070.

[33] Rasmussen KD, Helin K. Role of TET enzymes in DNA methylation, development, and cancer. Genes Dev 2016; 30: 733-50. https://doi.org/10.1101/gad.276568.115.

[34] Ke J, Bian X, Liu H, Li B, Huo R. Edaravone reduces oxidative stress and intestinal cell apoptosis after burn through upregulating miR-320 expression. Mol Med 2019; 25: 54.

[35] Jacob S, Herndon DN, Hawkins HK, Enkhbaatar P, Cox RA. Xanthine oxidase contributes to sustained airway epithelial oxidative stress after scald burn. Int J Burns Trauma 2017; 7: 98-106.

[36] Parihar A, Parihar MS, Milner S, Bhat S. Oxidative stress and anti-oxidative mobilization in burn injury. Burns J Int Soc Burn Inj 2008; 34: 6-17. https://doi.org/10.1016/j.burns.2007.04.009.

[37] Prabhakaran HS, Hu D, He W, Luo G, Liou YC. Mitochondrial dysfunction and mitophagy: crucial players in burn trauma and wound healing. Burns Trauma 2023; 11: tkad029. https://doi.org/10.1093/burnst/tkad029.

[38] Park E, Chung SW. ROS-mediated autophagy increases intracellular iron levels and ferroptosis by ferritin and transferrin receptor regulation. Cell Death Dis 2019; 10: 822. https://doi.org/10.1038/s41419-019-2064-5.

[39] Chen L, Zhang Z, Hoshino A, Zheng HD, Morley M, Arany Z, et al. NADPH production by the oxidative pentose-phosphate pathway supports folate metabolism. Nat Metab 2019; 1: 404-15. https://doi.org/10.1038/s42255-019-0043-x.

[40] Jiang LB, Cao L, Ma YQ, Chen Q, Liang Y, Yuan FL, et al. TIGAR mediates the inhibitory role of hypoxia on ROS production and apoptosis in rat nucleus pulposus cells. Osteoarthr Cartil 2018; 26: 138-48. https://doi.org/10.1016/j.joca.2017.10.007.

[41] Xiao Z, Deng S, Liu H, Wang R, Liu Y, Dai Z, et al. Glutamine deprivation induces ferroptosis in pancreatic cancer cells. Acta Biochim Biophys Sin 2023; 55: 1288-300. https://doi.org/10.3724/abbs.2023029.

[42] Liu MY, Li HM, Wang XY, Xia R, Li X, Ma YJ, et al. TIGAR drives colorectal cancer ferroptosis resistance through ROS/AMPK/SCD1 pathway. Free Radic Biol Med 2022; 182: 219-31. https://doi.org/10.1016/j.freeradbiomed.2022.03.002.

[43] Seo Y, Park SY, Kim HS, Nam JS. The hippo-YAP signaling as guardian in the pool of intestinal stem cells. Biomedicine 2020; 8: 560. https://doi.org/10.3390/biomedicines8120560.

[44] Moya IM, Halder G. Hippo-YAP/TAZ signalling in organ regeneration and regenerative medicine. Nat Rev Mol Cell Biol 2019; 20: 211-26. https://doi.org/10.1038/s41580-018-0086-y.

[45] Pobbati AV, Hong W. A combat with the YAP/TAZ-TEAD oncoproteins for cancer therapy. Theranostics 2020; 10: 3622-35. https://doi.org/10.7150/thno.40889.

[46] Cunningham R, Hansen CG. The hippo pathway in cancer: YAP/TAZ and TEAD as therapeutic targets in cancer. Clin Sci (Lond) 2022; 136: 197-222. https://doi.org/10.1042/CS20201474.

[47] Tang Y, Kwon H, Neel BA, Kasher-Meron M, Pessin JB, Yamada E, et al. The fructose-2,6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC. J Biol Chem 2018; 293: 7578-91. https://doi.org/10.1074/jbc.RA118.002727.

[48] Ganapathy-Kanniappan S, Geschwind JF. Tumor glycolysis as a target for cancer therapy: progress and prospects. Mol Cancer 2013; 12: 152. https://doi.org/10.1186/1476-4598-12-152.

[49] Li X, Yang Y, Zhang B, Lin X, Fu X, An Y, et al. Lactate metabolism in human health and disease. Signal Transduct Target Ther 2022; 7: 305. https://doi.org/10.1038/s41392-022-01151-3.

[50] Jafri F, Seong G, Jang T, Cimpeanu E, Poplawska M, Dutta D, et al. L-glutamine for sickle cell disease: more than reducing redox. Ann Hematol 2022; 101: 1645-54. https://doi.org/10.1007/s00277-022-04867-y.

[51] Kim JS, Martin MJ. REDOX REDUX? Glutamine, catabolism, and the urea-to-creatinine ratio as a novel nutritional metric. Crit Care Med 2022; 50: 1156-9. https://doi.org/10.1097/CCM.0000000000005520.

[52] Gong ZY, Yuan ZQ, Dong ZW, Peng YZ. Glutamine with probiotics attenuates intestinal inflammation and oxidative stress in a rat burn injury model through altered iNOS gene aberrant methylation. Am J Transl Res 2017; 9: 2535-47.

[53] Wu Q, Ni X. ROS-mediated DNA methylation pattern alterations in carcinogenesis. Curr Drug Targets 2015; 16: 13-9. https://doi.org/10.2174/1389450116666150113121054.

[54] Wang L, Mao B, Fan K, Sun R, Zhang J, Liang H, et al. ROS attenuates TET2-dependent ZO-1 epigenetic expression in cerebral vascular endothelial cells. Fluids Barriers CNS 2022; 19: 73. https://doi.org/10.1186/s12987-022-00370-8.