title: OGT-mediated O-GlcNAcylation promotes NF-κB activation and inflammation in acute pancreatitis.
abstract: OBJECTIVE
  Activation of the transcription factor κB (NF-κB) and secretion of pro-inflammatory mediators are major events in acute pancreatitis (AP). Recently, O-linked-N-acetylglucosamine (O-GlcNAc) modification, one type of posttranslational modifications, reportedly attunes NF-κB function. However, the expression of O-GlcNAc transferase (OGT), the enzyme responsible for O-GlcNAcylation of proteins, in AP, and the possible contribution of OGT-mediated O-GlcNAcylation to the NF-κB inflammatory activation in pancreatic acinar cells and to the AP progression have not been understood. This study focused on the effects and mechanisms of OGT-mediated O-GlcNAcylation during AP.
  
  
  METHODS
  An AP cell model was established with the caerulein-stimulated AR42 J rat pancreatic acinar cells. The secretion of pro-inflammatory cytokines TNF-α was detected by ELISA kits, and the production of NO was determined using the colorimetric Griess reaction. Expression of OGT was measured by RT-PCR and Western blot. Expression levels of RL2, phosphorylation of p65, total p65, IKKα were detected by Western blot. The NF-κB activity was evaluated by luciferase reporter gene assay. To determine the biological functions of OGT in caerulein-induced inflammatory response, RNA interference and PUGNAc, the inhibitor of O-GlcNAcase (OGA) was employed to regulate OGT expression in AR42 J cells.
  
  
  RESULTS
  Caerulein significantly up-regulated the expression of OGT, and increased the global protein O-GlcNAcylation level in AR42 J cells. Reduction of OGT by small interfering RNA (siRNA) inhibited caerulein-triggered inflammation, assessed by the production of pro-inflammatory mediators (TNF-α and NO). We also demonstrated that O-GlcNAcylation directly modified the NF-κB p65 subunit and its upstream activating kinases IKKα in AR42 J cells. Lowering O-GlcNAcylation by OGT knockdown attenuated p65 activating phosphorylation, nuclear translocation, NF-κB transcriptional activity and levels of NF-κB transcriptional targets TNF-α and NO; on the contrary, elevating O-GlcNAc through PUGNAc increased IKKα and p65 O-GlcNAcylation accompanied by increased p65 phosphorylation, activity and levels of TNF-α and NO in caerulein-treated cells.
  
  
  CONCLUSIONS
  Our results demonstrate for the first time that OGT-mediated O-GlcNAcylation promotes NF-κB signaling activation and inflammation in pancreatic acinar cells, which might promote the progression of AP.
authors.affiliation: Department of Pathogen Biology, Medical College, Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001, Jiangsu, People's Republic of China.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Department of Emergency, Affiliated Hospital of Nantong University, Nantong University, Nantong, 226001, Jiangsu, People's Republic of China.
authors.forename: Yongxia
authors.initials: Y
authors.lastname: Cai
authors.affiliation: Department of Emergency, Affiliated Hospital of Nantong University, Nantong University, Nantong, 226001, Jiangsu, People's Republic of China.
authors.forename: Minmin
authors.initials: M
authors.lastname: Chen
authors.affiliation: Department of Emergency, Affiliated Hospital of Nantong University, Nantong University, Nantong, 226001, Jiangsu, People's Republic of China.
authors.forename: Lili
authors.initials: L
authors.lastname: Gao
authors.affiliation: Department of Emergency, Affiliated Hospital of Nantong University, Nantong University, Nantong, 226001, Jiangsu, People's Republic of China. yanboshen123@163.com.
authors.forename: Yanbo
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Department of Emergency, Affiliated Hospital of Nantong University, Nantong University, Nantong, 226001, Jiangsu, People's Republic of China. zhongweihuang88@163.com.
authors.forename: Zhongwei
authors.initials: Z
authors.lastname: Huang
mesh_terms: D061354:Acinar Cells; D000208:Acute Disease; D000215:Acylation; D000818:Animals; D002460:Cell Line; D002108:Ceruletide / Q000494:pharmacology; D016207:Cytokines / Q000096:biosynthesis; D047468:Immunoprecipitation; D007249:Inflammation / Q000139:chemically induced* / Q000473:pathology; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D010195:Pancreatitis / Q000139:chemically induced* / Q000473:pathology; D034622:RNA Interference; D034741:RNA, Small Interfering; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D016207:Cytokines; D016328:NF-kappa B; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D002108:Ceruletide; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase
keywords: AR42 J; Acute pancreatitis; Inflammation; NF-κB; O-GLcNAc