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
View: Text | PDF
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

×

Figure 6

OGT deletion induces the expression of RIPK3 and MLKL in primary hepatocytes.

Options: View larger image (or click on image) Download as PowerPoint
OGT deletion induces the expression of RIPK3 and MLKL in primary hepatoc...
(A) Immunofluorescence images of primary hepatocytes stained with antibodies against RIPK3 and MLKL (green). The nucleus was stained with DAPI (blue). Fluorescence intensity was quantified with ImageJ (n = 30). (B) IHC stains of RIPK3 in liver sections from 4-week-old WT and OGT-LKO mice. (C and D) Western blotting of proteins from liver extracts (C) and primary hepatocytes (D) showing the expression levels of RIPK1, phosphorylated and total RIPK3, phosphorylated and total MLKL, and cleaved caspase 8 and caspase 3. Actin was used as loading control. (E) WT primary hepatocytes transfected with scrambled siRNA or OGT siRNA were treated with TNFA, cycloheximide (CHX), and z-VAD for 24 hours to induce necroptosis. Cell viability was determined with CellTiter-Glo luminescent cell viability assay kit (n = 3). Data were normalized to the DMSO-treated group. (F) Western blotting of RIPK3 and MLKL in primary hepatocytes transfected with scrambled siRNA or OGT siRNA and treated with TNFA, CHX, and z-VAD. Actin was used as loading control. (G) Liver weight to body weight ratio of saline or Nec-1s–treated mice. (H) ALT levels of saline or Nec-1s–treated mice. (I) H&E stains of saline or Nec-1s–treated mouse livers. All primary hepatocyte experiments were repeated at least 3 times with different cohorts of mice. 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; RIPK3, receptor-interacting protein kinase 3; MLKL, mixed lineage kinase domain-like; OGT-LKO, liver-specific OGT KO; RIPK1, receptor-interacting protein kinase 1; CHX, cycloheximide; Nec-1s, necrostatin 1 stable; ALT, alanine aminotransferase.