O-GlcNAc transferase suppresses necroptosis and liver fibrosis
Bichen Zhang, Min-Dian Li, Ruonan Yin, Yuyang Liu, Yunfan Yang, Kisha A. Mitchell-Richards, Jin Hyun Nam, Rui Li, Li Wang, Yasuko Iwakiri, Dongjun Chung, Marie E. Robert, Barbara E. Ehrlich, Anton M. Bennett, Jun Yu, Michael H. Nathanson, Xiaoyong Yang
Bichen Zhang, Min-Dian Li, Ruonan Yin, Yuyang Liu, Yunfan Yang, Kisha A. Mitchell-Richards, Jin Hyun Nam, Rui Li, Li Wang, Yasuko Iwakiri, Dongjun Chung, Marie E. Robert, Barbara E. Ehrlich, Anton M. Bennett, Jun Yu, Michael H. Nathanson, Xiaoyong Yang
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Research Article Hepatology Inflammation

O-GlcNAc transferase suppresses necroptosis and liver fibrosis

Abstract

Worldwide, over a billion people suffer from chronic liver diseases, which often lead to fibrosis and then cirrhosis. Treatments for fibrosis remain experimental, in part because no unifying mechanism has been identified that initiates liver fibrosis. Necroptosis has been implicated in multiple liver diseases. Here, we report that O-linked β-N-acetylglucosamine (O-GlcNAc) modification protects against hepatocyte necroptosis and initiation of liver fibrosis. Decreased O-GlcNAc levels were seen in patients with alcoholic liver cirrhosis and in mice with ethanol-induced liver injury. Liver-specific O-GlcNAc transferase–KO (OGT-LKO) mice exhibited hepatomegaly and ballooning degeneration at an early age and progressed to liver fibrosis and portal inflammation by 10 weeks of age. OGT-deficient hepatocytes underwent excessive necroptosis and exhibited elevated protein expression levels of receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), which are key mediators of necroptosis. Furthermore, glycosylation of RIPK3 by OGT is associated with reduced RIPK3 protein stability. Taken together, these findings identify OGT as a key suppressor of hepatocyte necroptosis, and OGT-LKO mice may serve as an effective spontaneous genetic model of liver fibrosis.

Authors

Bichen Zhang, Min-Dian Li, Ruonan Yin, Yuyang Liu, Yunfan Yang, Kisha A. Mitchell-Richards, Jin Hyun Nam, Rui Li, Li Wang, Yasuko Iwakiri, Dongjun Chung, Marie E. Robert, Barbara E. Ehrlich, Anton M. Bennett, Jun Yu, Michael H. Nathanson, Xiaoyong Yang

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Figure 5

Loss of OGT in hepatocytes leads to excessive necroptosis.

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Loss of OGT in hepatocytes leads to excessive necroptosis. (A) Primary h...
(A) Primary hepatocyte cell size quantified under light microscopy (n = 50). (B) Primary hepatocyte viability determined by Trypan blue staining (n = 3). (C) ATP content determined by the CellTiter-Glo luminescent cell viability assay kit (n = 3). Data were normalized to cell number. (D) Flow cytometry analysis of annexin V–propidium iodine (PI) double-stained primary hepatocytes. Data were normalized to total cell number (n = 3). (E) Western blotting of phosphorylated MLKL in primary hepatocytes. (F) Primary hepatocyte cell viability. z-VAD is a pan-caspase inhibitor (n = 4). (G and H) Images and quantification of MitoSox stains of primary hepatocytes. Fluorescence intensity was quantified with ImageJ (n = 20). Scale bar: 50 μm. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired Student’s t test. OGT, O-GlcNAc transferase; GSK, GSK-872, RIPK3 kinase inhibitor; RIPK3, receptor-interacting protein kinase 3; MLKL, mixed lineage kinase domain-like.