O-GlcNAc transferase suppresses necroptosis and liver fibrosis.

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 ß-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.

Chemopreventive Efficacy of the Cyclooxygenase-2 (Cox-2) Inhibitor, Celecoxib, Is Predicted by Adenoma Expression of Cox-2 and 15-PGDH.

Background: The Adenoma Prevention with Celecoxib (APC) Trial showed that cyclooxygenase-2 (Cox-2) inhibitor, celecoxib, decreased adenoma development in patients at high risk for colorectal cancer. A prospectively planned analysis of the APC Trial tested the hypothesis that expression of target enzymes in adenomas removed before beginning study treatment would identify individuals at high risk of adenoma development, and/or predict response to Cox-2 inhibition.Methods: Pre-treatment adenomas were examined using immunohistochemistry to assess expression of Cox-2 (high vs. low) and 15-prostaglandin dehydrogenase (15-PGDH, presence vs. loss). The Mantel-Cox test evaluated whether these markers predicted benefit from celecoxib for reduction of adenoma detection.Results: Patients whose pre-treatment adenomas demonstrated elevated Cox-2 achieved the greatest adenoma reduction with celecoxib treatment [RR, 0.37; 95% confidence interval (CI), 0.22-0.61; P = 0.0001]. This reduction was less in the low Cox-2 category (RR, 0.64; 95% CI, 0.56-0.73). Patients whose pre-treatment adenomas showed 15-PGDH loss had a similar treatment-associated reduction in adenoma detection (RR, 0.60; 95% CI, 0.52-0.69; P < 0.0001). In contrast, patients with intact tumor 15-PGDH expression did not significantly benefit from celecoxib (RR, 0.73; 95% CI, 0.47-1.12; P = 0.15). However, subset analysis suggested that this lack of response to celecoxib was confined to those patients with 15-PGDH intact tumors who were also using cardioprotective aspirin.Conclusions: The expression of Cox-2 and 15-PGDH in pre-treatment adenomas provides predictive information in patients treated with celecoxib for prevention of colorectal adenomas.Impact: The results of this study show that Cox-2 and 15-PGDH are characteristics of colorectal adenomas that may be used to predict nonsteroidal anti-inflammatory drug chemoprevention efficacy. Cancer Epidemiol Biomarkers Prev; 27(7); 728-36. ©2018 AACR.

Nonalcoholic fatty liver disease impairs expression of the type II inositol 1,4,5-trisphosphate receptor.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease worldwide. It may result in several types of liver problems, including impaired liver regeneration (LR), but the mechanism for this is unknown. Because LR depends on calcium signaling, we examined the effects of NAFLD on expression of the type II inositol 1,4,5-trisphosphate receptor (ITPR2), the principle calcium release channel in hepatocytes. ITPR2 promoter activity was measured in Huh7 and HepG2 cells. ITPR2 and c-Jun protein levels were evaluated in Huh7 cells, in liver tissue from a rat model of NAFLD, and in liver biopsy specimens of patients with simple steatosis and nonalcoholic steatohepatitis (NASH). LR was assessed in wild-type and Itpr2 knockout (Itpr2-/- ) mice following 67% hepatectomy. Cell proliferation was examined in ITPR2-knockout HepG2 cells generated by the CRISPR/Cas9 system. c-Jun dose dependently decreased activity of the human ITPR2 promoter. c-Jun expression was increased and ITPR2 was decreased in fat-loaded Huh7 cells and in livers of rats fed a high-fat, high-fructose diet. Overexpression of c-Jun reduced protein and mRNA expression of ITPR2 in Huh7 cells, whereas knockdown of c-Jun prevented the decrease of ITPR2 in fat-loaded Huh7 cells. ITPR2 expression was decreased and c-Jun was increased in liver biopsies of patients with steatosis and NASH compared to controls. ITPR2-knockout cells exhibited less nuclear calcium signaling and cell proliferation than control cells. LR assessed by Ki-67 and proliferating cell nuclear antigen was markedly decreased in Itpr2-/- mice. Conclusion: Fatty liver induces a c-Jun-mediated decrease in ITPR2 in hepatocytes. This may account for the impaired LR that occurs in NAFLD. (Hepatology 2018;67:560-574).

Nuclear Factor, Erythroid 2-Like 2 Regulates Expression of Type 3 Inositol 1,4,5-Trisphosphate Receptor and Calcium Signaling in Cholangiocytes.

BACKGROUND & AIMS: Most cholestatic disorders are caused by defects in cholangiocytes. The type 3 isoform of the inositol 1,4,5-trisphosphate receptor (ITPR3) is the most abundant intracellular calcium release channel in cholangiocytes. ITPR3 is required for bicarbonate secretion by bile ducts, and its expression is reduced in intrahepatic bile ducts of patients with cholestatic disorders. We investigated whether the nuclear factor, erythroid 2-like 2 (NFE2L2 or NRF2), which is sensitive to oxidative stress, regulates expression of ITPR3. METHODS: The activity of the ITPR3 promoter was measured in normal human cholangiocyte (NHC) cells and primary mouse cholangiocytes. Levels of ITPR3 protein and messenger RNA were examined by immunoblot and polymerase chain reaction analyses, respectively. ITPR3 activity was determined by measuring calcium signaling in normal human cholangiocyte cells and secretion in isolated bile duct units. Levels of NRF2 were measured in liver tissues from rats with cholestasis (induced by administration of α-napthylisothiocyanate) and from patients with biliary diseases. RESULTS: We identified a musculo-aponeurotic fibrosarcoma recognition element in the promoter of ITPR3 that bound NRF2 directly in NHC cells and mouse cholangiocytes. Increasing binding of NRF2 at this site resulted in chromatin remodeling that reduced promoter activity. Mutant forms of the musculo-aponeurotic fibrosarcoma recognition element did not bind NRF2. Activation of NRF2 with quercetin or by oxidative stress reduced expression of ITPR3 and calcium signaling in NHC cells; quercetin also reduced secretion by bile duct units isolated from rats. Knockdown of NRF2 with small interfering RNAs restored expression and function of ITPR3 in NHC cells incubated with quercetin. Bile ducts from rats with cholestasis and patients with cholangiopathic disorders expressed higher levels of NRF2 and lower levels of ITPR3 than ducts from control rats or patients with other liver disorders. CONCLUSIONS: The transcription factor NRF2 binds to the promoter of ITPR3 to inhibit its expression in cholangiocytes, leading to reduced calcium signaling and bile duct secretion. This could be a mechanism by which oxidative stress inhibits these processes and contributes to cholangiopathies.