CYLD Regulates T Cell Metabolism and Mitochondrial Autophagy through LKB1/AMPKα Pathway

Abstract

The deubiquitinating enzyme cylindromatosis (CYLD) plays a fundamental role in regulating T cell development and activation. Previous studies have shown that CYLD is associated with autophagy, while AMP activated protein kinase (AMPK) pathway regulates the development of autophagy and affects cell metabolism. However, the mechanism by which CYLD affects autophagy and whether it affects the downstream metabolism of AMPKα remains unclear. In this study, we used the CYLD gene knockout model in Jurkat cells to investigate the mechanism of CYLD and autophagy and its relationship with cellular metabolism. The results show that CYLD deletion promotes autophagy through AMPKα/mTOR/ULK1 signaling pathway, promotes mitochondrial autophagy to improve mitochondrial function and attenuates cell lipid metabolism in Jurkat cells.

Keywords

CYLD / AMPK / Autophagy / Metabolism / T cells / Mitochondria

Supplementary Materials

The following supporting information can be found at: https://www.sciepublish.com/article/pii/399. Figure S1. Two groups of cells in basic conditions. (A). CCK-8 assay to determine the proliferation of Jurkat cells and CYLD knock Jurkat T cells. (B) The image of Jurkat cells and CYLD knock Jurkat T cells (20x). (C) The protein level of LC3 were evaluated by immunoblotting analysis in basal conditions. (D) The mRNA expression level of GAPDH by RTPCR. The data shown represent three independent experiments with similar results; Figure S2. The protein level of CPT1A; Figure S3. CYLD deficiency decreased inflammatory factors. (A,B). The level of inflammatory factors in the supernatant was measured by ELISA analysis. The data shown represent three independent experiments with similar results.

Author Contributions

H.W. and L.Y. wrote the manuscript; H.W., L.Y., and J.Y. contributed conception and design of the study. L.Y., G.G., J.G. and Y.Z. contributed to the experiments. Y.L. generated and supplied CYLD^−/− Jurkat cells. Y.N. provided expertise and advice. H.W. oversaw the project. All authors contributed to the manuscript revision, and have read and approved the submitted version.

Ethics Statement

Not applicable.

Informed Consent Statement.

Not applicable.

Data Availability Statement

Data will be made available on request.

Funding

This work was funded by the National Natural Science Foundation of China (Grant No. U1804190) and the 111Project (No. D20036).

Declaration of Competing Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

[1] Haas R, Cucchi D, Smith J, Pucino V, Macdougall CE, Mauro C. Intermediates of Metabolism: From Bystanders to Signalling Molecules. Trends Biochem. Sci. 2016, 41, 460-471.

[2] Liu H, Zeng L, Pan M, Huang L, Li H, Liu M, et al. Bcl-3 regulates T cell function through energy metabolism. BMC Immunol. 2023, 24, 35.

[3] Zhang X, Liu J, Cao X. Metabolic control of T-cell immunity via epigenetic mechanisms. Cell Mol. Immunol. 2018, 15, 203-205.

[4] Wang R, Green DR. Metabolic checkpoints in activated T cells. Nat. Immunol. 2012, 13, 907-915.

[5] Mehta MM, Weinberg SE, Chandel NS. Mitochondrial control of immunity: beyond ATP. Nat. Rev. Immunol. 2017, 17, 608-620.

[6] Buck MD, O’Sullivan D, Geltink RIK, Curtis JD, Chang CH, Sanin DE, et al. Mitochondrial Dynamics Controls T Cell Fate through Metabolic Programming. Cell 2016, 166, 63-76.

[7] Chiticariu E, Regamey A, Huber M, Hohl D. CENPV Is a CYLD-Interacting Molecule Regulating Ciliary Acetylated alpha-Tubulin. J. Investig. Dermatol. 2020, 140, 66-74 e4.

[8] Mathis JB, Lai Y, Qu C, Janicki SJ, Cui T.CYLD-mediated signaling and diseases. Curr. Drug Targets 2015, 16, 284-294.

[9] Reiley WW, Zhang M, Jin W, Losiewicz M, Donohue KB, Norbury CC, et al. Regulation of T cell development by the deubiquitinating enzyme CYLD. Nat. Immunol. 2006, 7, 411-417.

[10] Lee BC, Miyata M, Lim JH, Li JD.Deubiquitinase CYLD acts as a negative regulator for bacterium NTHi-induced inflammation by suppressing K63-linked ubiquitination of MyD88. Proc. Natl. Acad. Sci. USA 2016, 113, E165-E171.

[11] Zhang LM, Zhou JJ, Luo CL. CYLD suppression enhances the pro-inflammatory effects and hyperproliferation of rheumatoid arthritis fibroblast-like synoviocytes by enhancing NF-kappaB activation. Arthritis Res. Ther. 2018, 20, 219.

[12] Lork M, Verhelst K, Beyaert R. CYLD, A20 and OTULIN deubiquitinases in NF-kappaB signaling and cell death: so similar, yet so different. Cell Death Differ. 2017, 24, 1172-1183.

[13] Massoumi R, Chmielarska K, Hennecke K, Pfeifer A, Fässler R. Cyld inhibits tumor cell proliferation by blocking Bcl-3-dependent NF-kappaB signaling. Cell 2006, 125, 665-677.

[14] Fernández-Majada V, Welz PS, Ermolaeva MA, Schell M, Adam A, Dietlein F, et al. The tumour suppressor CYLD regulates the p53 DNA damage response. Nat. Commun. 2016, 7, 12508.

[15] Hellerbrand C, Bumes E, Bataille F, Dietmaier W, Massoumi R, Bosserhoff AK. Reduced expression of CYLD in human colon and hepatocellular carcinomas. Carcinogenesis 2007, 28, 21-27.

[16] Pseftogas A, Xanthopoulos K, Siasiaridis A, Poutahidis T, Gonidas C, Tsingotjidou A, et al. Inactivation of the Tumor Suppressor CYLD Sensitizes Mice to Breast Cancer Development. Anticancer Res. 2024, 44, 1885-1894.

[17] Wang J, Niu Z, Shi Y, Gao C, Wang X, Han J, et al. Bcl-3, induced by Tax and HTLV-1, inhibits NF-kappaB activation and promotes autophagy. Cell Signal 2013, 25, 2797-2804.

[18] Mizushima N, Yoshimori T, Levine B.Methods in mammalian autophagy research. Cell 2010, 140, 313-326.

[19] Yin L, Liu S, Li C, Ding S, Bi D, Niu Z, et al. CYLD downregulates Living and synergistically improves gemcitabine chemosensitivity and decreases migratory/invasive potential in bladder cancer: the effect is autophagy-associated. Tumour Biol. 2016, 37, 12731-12742.

[20] Bonapace L, Bornhauser BC, Schmitz M, Cario G, Ziegler U, Niggli FK, et al. Induction of autophagy-dependent necroptosis is required for childhood acute lymphoblastic leukemia cells to overcome glucocorticoid resistance. J. Clin. Investig. 2010, 120, 1310-1323.

[21] Kim J, Kundu M, Viollet B, Guan KL.AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol. 2011, 13, 132-141.

[22] Li X, Liu J, Lu Q, Ren D, Sun X, Rousselle T, et al. AMPK: A therapeutic target of heart failure-not only metabolism regulation. Biosci. Rep. 2019, 39, BSR20181767.

[23] Hou Y, Fu J, Sun S, Jin Y, Wang X, Zhang L. BDE-209 induces autophagy and apoptosis via IRE1alpha/Akt/mTOR signaling pathway in human umbilical vein endothelial cells. Environ. Pollut. 2019, 253, 429-438.

[24] Lee SB, Kim JJ, Han SA, Fan Y, Guo LS, Aziz K, Nowsheen S, et al. The AMPK-Parkin axis negatively regulates necroptosis and tumorigenesis by inhibiting the necrosome. Nat. Cell. Biol. 2019, 21, 940-951.

[25] Chen X, Luo Y, Wang M, Sun L, Huang K, Li Y, et al. Wuhu Decoction regulates dendritic cell autophagy in the treatment of respiratory syncytial virus (RSV)-induced mouse asthma by AMPK/ULK1 signaling pathway. Med. Sci. Monit. 2019, 25, 5389-5400.

[26] Guan J, Lin H, Xie M, Huang M, Zhang D, Ma S, et al. Higenamine exerts an antispasmodic effect on cold-induced vasoconstriction by regulating the PI3K/Akt, ROS/alpha2C-AR and PTK9 pathways independently of the AMPK/eNOS/NO axis. Exp. Ther. Med. 2019, 18, 1299-1308.

[27] Mohsin AA, Chen Q, Quan N, Rousselle T, Maceyka MW, Samidurai A, et al. Mitochondrial complex I inhibition by metformin limits reperfusion injury. J. Pharmacol. Exp. Ther. 2019, 369, 282-290.

[28] Ross T, Szczepanek K, Bowler E, Hu Y, Larner A, Lesnefsky EJ, et al. Reverse electron flow-mediated ROS generation in ischemia-damaged mitochondria: role of complex I inhibition vs. depolarization of inner mitochondrial membrane. Biochim. Biophys. Acta 2013, 1830, 4537-4542.

[29] Li L, Pan R, Li R, Niemann B, Aurich AC, Chen Y, et al. Mitochondrial biogenesis and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) deacetylation by physical activity: intact adipocytokine signaling is required. Diabetes 2011, 60, 157-167.

[30] Herzig S, Shaw RJ. AMPK: guardian of metabolism and mitochondrial homeostasis. Nat. Rev. Mol. Cell Biol. 2018, 19, 121-135.

[31] Seo MS, Kim JH, Kim HJ, Chang KC, Park SW. Honokiol activates the LKB1-AMPK signalling pathway and attenuates the lipid accumulation in hepatocytes. Toxicol. Appl. Pharmacol. 2015, 284, 113-124.

[32] Deng J, Guo Y, Yuan F, Chen S, Yin H, Jiang X, et al. Autophagy inhibition prevents glucocorticoid-increased adiposity via suppressing BAT whitening. Autophagy 2020, 16, 451-465.

[33] Deng B, Wang J, Yang T, Deng Z, Yuan J, Zhang B, et al. TNF and IFNgamma-induced cell death requires IRF1 and ELAVL1 to promote CASP8 expression. J. Cell Biol. 2024, 223, e202305026.

[34] Mukherjee S, Kumar R, Tsakem Lenou E, Basrur V, Kontoyiannis DL, Ioakeimidis F, et al. Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation. Nat. Immunol. 2020, 21, 626-635.

[35] Thuille N, Wachowicz K, Hermann-Kleiter N, Kaminski S, Fresser F, Lutz-Nicoladoni C, et al. PKCtheta/beta and CYLD are antagonistic partners in the NF-kappaB and NFAT transactivation pathways in primary mouse CD3+ T lymphocytes. PLoS ONE 2013, 8, e53709.

[36] DeVorkin L, Pavey N, Carleton G, Comber A, Ho C, Lim J, et al. Autophagy regulation of metabolism is required for CD8⁺ T cell anti-tumor immunity. Cell Rep. 2019, 27, 502-513 e5.

[37] Dowling SD, Macian F.Autophagy and T cell metabolism. Cancer Lett. 2018, 419, 20-26.

[38] Mocholi E, Dowling SD, Botbol Y, Gruber RC, Ray AK, Vastert S, et al. Autophagy is a tolerance-avoidance mechanism that modulates TCR-mediated signalling and cell metabolism to prevent induction of T cell anergy. Cell Rep. 2018, 24, 1136-1150.

[39] Yang G, Song W, Postoak JL, Chen J, Martinez J, Zhang J, et al. Autophagy-related protein PIK3C3/VPS34 controls T cell metabolism and function. Autophagy 2020, 1, 1-12.

[40] Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, Mair W, et al. Phosphorylation of ULK 1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 2011, 331, 456-561.

[41] Foretz M, Guigas B, Viollet B. Understanding the glucoregulatory mechanisms of metformin in type 2 diabetes mellitus. Nat. Rev. Endocrinol. 2019, 15, 569-589.