Ubc9-mediated SUMOylation of Ninj1 alleviates inflammatory responses in hepatic ischaemia/reperfusion injury

Abstract

Background: Hepatic ischaemia/reperfusion (I/R) injury poses a common clinical dilemma encountered during liver transplantation (LT), characterised by substantial cellular death and inflammation reactions. Ubc9, the sole E2 conjugating enzyme of SUMOylation, has long been recognised to regulate diverse biological and pathological processes. However, its impact on I/R-induced liver damage is yet to be elucidated.

Methods: The expression levels of UBC9 in patients undergoing LT were analysed. Hepatocyte-specific Ubc9-deficient or transgenic mice were utilised in an in vivo model of hepatic I/R, alongside in vitro experiments that employed hypoxia/reoxygenation stimulation. The investigation focused on Ubc9's role in liver damage due to I/R and the underlying mechanisms through a range of phenotypic analyses and biological techniques.

Results: Herein, we found that hepatic tissues from patients with LT are featured by a significant downregulation of UBC9 expression. Studies in 68 donor hepatic biopsies further demonstrated a negative correlation between UBC9 expression and liver injury in patients with LT. Similarly, murine liver I/R was coupled with an obvious decrease in Ubc9 expression. Hepatocyte deficient in Ubc9 exacerbated liver injury in liver I/R, while Ubc9-overexpression showed the opposite phenotype. Mechanistically, Ubc9-mediated SUMOylation of Ninj1 at lysine K103 inhibited its membrane localisation and damage-associated molecular patterns (DAMPs) release in hepatocytes, subsequently inhibited nuclear factor-kappa B (NF-κB) signalling in macrophages and curtailing inflammatory cytokines production.

Conclusions: These findings further suggest that Ubc9-mediated SUMOylation of Ninj1 at lysine K103 may represent a potential therapeutic strategy for safeguarding the liver against I/R injury in clinical settings.

Key points:

Keywords

hepatic ischaemia/reperfusion / Ninj1 / SUMOylation / Ubc9

References

[1] Hirao H, Nakamura K, Kupiec-Weglinski JW. Liver ischaemia‒reperfusion injury: a new understanding of the role of innate immunity. Nat Rev Gastroenterol Hepatol. 2022; 19(4): 239-256.

[2] Lin CS, He MQ, An MY, et al. Ultra-small copper-based multienzyme-like nanoparticles protect against hepatic ischemia‒reperfusion injury through scavenging reactive oxygen species in mice. Small. 2024; 20(51):e2403313.

[3] Liu H, Man K. New insights in mechanisms and therapeutics for short- and long-term impacts of hepatic ischemia reperfusion injury post liver transplantation. Int J Mol Sci. 2021; 22(15): 8210.

[4] Nakamura K, Zhang M, Kageyama S, et al. Macrophage heme oxygenase-1-SIRT1-p53 axis regulates sterile inflammation in liver ischemia‒reperfusion injury. J Hepatol. 2017; 67(6): 1232-1242.

[5] Dar WA, Sullivan E, Bynon JS, Eltzschig H, Ju C. Ischaemia reperfusion injury in liver transplantation: cellular and molecular mechanisms. Liver Int. 2019; 39(5): 788-801.

[6] Zhang Q, Chen Z, Li J, et al. The deubiquitinase OTUD1 stabilizes NRF2 to alleviate hepatic ischemia/reperfusion injury. Redox Biol. 2024; 75:103287.

[7] Zhang S, Sun Z, Chen Z, et al. Endothelial YAP/TEAD1-CXCL17 signaling recruits myeloid-derived suppressor cells against liver ischemia‒reperfusion injury. Hepatology. 2025; 81(3): 888-902.

[8] Rao J, Qiu J, Ni M, et al. Macrophage nuclear factor erythroid 2-related factor 2 deficiency promotes innate immune activation by tissue inhibitor of metalloproteinase 3-mediated RhoA/ROCK pathway in the ischemic liver. Hepatology. 2022; 75(6): 1429-1445.

[9] Ni M, Zhang J, Sosa R, et al. T-cell immunoglobulin and mucin domain-containing protein-4 is critical for Kupffer cell homeostatic function in the activation and resolution of liver ischemia reperfusion injury. Hepatology. 2021; 74(4): 2118-2132.

[10] Chang HM, Yeh ETH. SUMO: from bench to bedside. Physiol Rev. 2020; 100(4): 1599-1619.

[11] Vertegaal ACO. Signalling mechanisms and cellular functions of SUMO. Nat Rev Mol Cell Biol. 2022; 23(11): 715-731.

[12] Talamillo A, Barroso-Gomila O, Giordano I, et al. The role of SUMOylation during development. Biochem Soc Trans. 2020; 48(2): 463-478.

[13] Gallardo-Chamizo F, González-Prieto R, Jafari V, Luna-Peláez N, Vertegaal ACO, García-Domínguez M. SUMO2/3 modification of transcription-associated proteins controls cell viability in response to oxygen and glucose deprivation-mediated stress. Cell Death Discov. 2025; 11(1): 230.

[14] Claessens LA, Vertegaal ACO. SUMO proteases: from cellular functions to disease. Trends Cell Biol. 2024; 34(11): 901-912.

[15] Karhausen J, Bernstock JD, Johnson KR, et al. Ubc9 overexpression and SUMO1 deficiency blunt inflammation after intestinal ischemia/reperfusion. Lab Invest. 2018; 98(6): 799-813.

[16] Hewitt WM, Lountos GT, Zlotkowski K, et al. Insights into the allosteric inhibition of the SUMO E2 enzyme Ubc9. Angew Chem Int Ed Engl. 2016; 55(19): 5703-5707.

[17] Xiao Q, Chen XH, Jiang RC, et al. Ubc9 attenuates myocardial ischemic injury through accelerating autophagic flux. Front Pharmacol. 2020; 11:561306.

[18] Kayagaki N, Kornfeld OS, Lee BL, et al. NINJ1 mediates plasma membrane rupture during lytic cell death. Nature. 2021; 591(7848): 131-136.

[19] Degen M, Santos JC, Pluhackova K, et al. Structural basis of NINJ1-mediated plasma membrane rupture in cell death. Nature. 2023; 618(7967): 1065-1071.

[20] David L, Borges JP, Hollingsworth LR, et al. NINJ1 mediates plasma membrane rupture by cutting and releasing membrane disks. Cell. 2024; 187(9): 2224-2235.e2216.

[21] Kim MW, Kang JH, Jung HJ, et al. Deficiency of Ninjurin1 attenuates LPS/D-galactosamine-induced acute liver failure by reducing TNF-α-induced apoptosis in hepatocytes. J Cell Mol Med. 2022; 26(20): 5122-5134.

[22] Hu Y, Zhan F, Wang Y, et al. The Ninj1/Dusp1 axis contributes to liver ischemia reperfusion injury by regulating macrophage activation and neutrophil infiltration. Cell Mol Gastroenterol Hepatol. 2023; 15(5): 1071-1084.

[23] Xie H, Gao Z, Liu X, et al. Ubc9-mediated SUMOylation of RPL3, an unappreciated mechanism against hepatocyte senescence by repressing the DHX9-p16 axis. Adv Sci. 2026;13:e10240.

[24] Toda G, Yamauchi T, Kadowaki T, Ueki K. Preparation and culture of bone marrow-derived macrophages from mice for functional analysis. STAR Protoc. 2021; 2(1):100246.

[25] Mohar I, Brempelis KJ, Murray SA, Ebrahimkhani MR, Crispe IN. Isolation of non-parenchymal cells from the mouse liver. Methods Mol Biol. 2015; 1325: 3-17.

[26] Gong Y, You Q, Yuan X, et al. Mesenchymal stem cell-derived extracellular vesicles attenuate ferroptosis in aged hepatic ischemia/reperfusion injury by transferring miR-1275. Redox Biol. 2025; 81:103556.

[27] Li L, Huang K, Zhao H, Chen B, Ye Q, Yue J. CDK1-PLK1/SGOL2/ANLN pathway mediating abnormal cell division in cell cycle may be a critical process in hepatocellular carcinoma. Cell Cycle. 2020; 19(10): 1236-1252.

[28] Kayagaki N, Stowe IB, Alegre K, et al. Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury. Nature. 2023; 618(7967): 1072-1077.

[29] Li J, Zhao J, Xu M, et al. Blocking GSDMD processing in innate immune cells but not in hepatocytes protects hepatic ischemia‒reperfusion injury. Cell Death Dis. 2020; 11(4): 244.

[30] Nakamura K, Kageyama S, Kaldas FM, et al. Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury. J Clin Invest. 2020; 130(5): 2689-2704.

[31] Huang H, Tohme S, Al-Khafaji AB, et al. Damage-associated molecular pattern-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury. Hepatology. 2015; 62(2): 600-614.

[32] Tsung A, Klune JR, Zhang X, et al. HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and calcium-mediated signaling. J Exp Med. 2007; 204(12): 2913-2923.

[33] Evankovich J, Cho SW, Zhang R, et al. High mobility group box 1 release from hepatocytes during ischemia and reperfusion injury is mediated by decreased histone deacetylase activity. J Biol Chem. 2010; 285(51): 39888-39897.

[34] Li XC. NINJ1 powers membrane rupture during inflammatory cell death. Am J Transplant. 2023; 23(8): 1090-1091.

[35] Zhu Y, Xiao F, Wang Y, et al. NINJ1 regulates plasma membrane fragility under mechanical strain. Nature. 2025; 644(8078): 1088-1096.

[36] Li Y, Tang Y, Yan X, et al. CARD9 protein SUMOylation regulates HOXB5 nuclear translocation and Parkin-mediated mitophagy in myocardial I/R injury. J Cell Mol Med. 2024; 28(21):e70195.

[37] Xue S, Zeng J, Hao J, et al. SENP2-mediated deSUMOylation of NCOA4 protects against ferritinophagy-dependent ferroptosis in myocardial ischemia‒reperfusion injury. Autophagy. 2025; 21: 2367-2384.

[38] Xiao J, Sun F, Wang YN, et al. UBC9 deficiency enhances immunostimulatory macrophage activation and subsequent antitumor T cell response in prostate cancer. J Clin Invest. 2023; 133(4):e158352.

[39] Chen C, Zheng H, Luo Y, et al. SUMOylation promotes extracellular vesicle-mediated transmission of lncRNA ELNAT1 and lymph node metastasis in bladder cancer. J Clin Invest. 2021; 131(8):e146431.

[40] Yan L, Zhang T, Wang K, et al. SENP1 prevents steatohepatitis by suppressing RIPK1-driven apoptosis and inflammation. Nat Commun. 2022; 13(1): 7153.

[41] Nace GW, Huang H, Klune JR, et al. Cellular-specific role of toll-like receptor 4 in hepatic ischemia‒reperfusion injury in mice. Hepatology. 2013; 58(1): 374-387.

[42] Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature. 2002; 418(6894): 191-195.

[43] Li Q, Lan P. Activation of immune signals during organ transplantation. Signal Transduct Target Ther. 2023; 8(1): 110.

[44] Dhupar R, Klune JR, Evankovich J, et al. Interferon regulatory factor 1 mediates acetylation and release of high mobility group box 1 from hepatocytes during murine liver ischemia‒reperfusion injury. Shock. 2011; 35(3): 293-301.

[45] Hernandez C, Huebener P, Pradere JP, Antoine DJ, Friedman RA, Schwabe RF. HMGB1 links chronic liver injury to progenitor responses and hepatocarcinogenesis. J Clin Invest. 2019; 129(4): 1803.

[46] Kadono K, Uchida Y, Hirao H, et al. Thrombomodulin attenuates inflammatory damage due to liver ischemia and reperfusion injury in mice in Toll-like receptor 4-dependent manner. Am J Transplant. 2017; 17(1): 69-80.

[47] Sugihara M, Sadamori H, Nishibori M, et al. Anti-high mobility group box 1 monoclonal antibody improves ischemia/reperfusion injury and mode of liver regeneration after partial hepatectomy. Am J Surg. 2016; 211(1): 179-188.

[48] Zhao QH, Zhang YT, Wen K, et al. Myeloid but not hepatocytic CD38 is a key driver for hepatic ischemia/reperfusion injury. Signal Transduct Target Ther. 2025; 10(1): 150.

[49] Franz S, Ertel A, Engel KM, Simon JC, Saalbach A. Overexpression of S100A9 in obesity impairs macrophage differentiation via TLR4‒NFkB-signaling worsening inflammation and wound healing. Theranostics. 2022; 12(4): 1659-1682.

[50] Kayagaki N, Webster JD, Newton K. Control of cell death in health and disease. Annu Rev Pathol. 2024; 19: 157-180.

[51] Araki T, Milbrandt J. Ninjurin, a novel adhesion molecule, is induced by nerve injury and promotes axonal growth. Neuron. 1996; 17(2): 353-361.

[52] Yang HJ, Zhang J, Yan W, et al. Ninjurin 1 has two opposing functions in tumorigenesis in a p53-dependent manner. Proc Natl Acad Sci U S A. 2017; 114(43): 11500-11505.

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2026 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.