Zhang Dongmei, Cai Yongxia, Chen Minmin, Gao Lili, Shen Yanbo, Huang Zhongwei
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.
Department of Emergency, Affiliated Hospital of Nantong University, Nantong University, Nantong, 226001, Jiangsu, People's Republic of China.
Inflamm Res. 2015 Dec;64(12):943-52. doi: 10.1007/s00011-015-0877-y. Epub 2015 Sep 25.
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.
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.
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.
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.
转录因子κB（NF-κB）的激活和促炎介质的分泌是急性胰腺炎（AP）的主要事件。最近，据报道，O-连接的N-乙酰葡糖胺（O-GlcNAc）修饰作为一种翻译后修饰，可调节NF-κB功能。然而，负责蛋白质O-GlcNAc化的酶——O-GlcNAc转移酶（OGT）在AP中的表达，以及OGT介导的O-GlcNAcylation对胰腺腺泡细胞中NF-κB炎症激活和AP进展的可能作用尚不清楚。本研究聚焦于AP过程中OGT介导的O-GlcNAcylation的作用及机制。
用雨蛙素刺激AR42J大鼠胰腺腺泡细胞建立AP细胞模型。采用ELISA试剂盒检测促炎细胞因子TNF-α的分泌，用比色法Griess反应测定NO的产生。通过RT-PCR和Western blot检测OGT的表达。用Western blot检测RL2的表达水平、p65的磷酸化、总p65、IKKα。通过荧光素酶报告基因测定评估NF-κB活性。为了确定OGT在雨蛙素诱导的炎症反应中的生物学功能，采用RNA干扰和O-GlcNAcase（OGA）抑制剂PUGNAc来调节AR42J细胞中OGT的表达。
雨蛙素显著上调AR42J细胞中OGT的表达，并增加整体蛋白O-GlcNAcylation水平。通过小干扰RNA（siRNA）降低OGT可抑制雨蛙素引发的炎症，这通过促炎介质（TNF-α和NO）的产生来评估。我们还证明，O-GlcNAcylation直接修饰AR42J细胞中的NF-κB p65亚基及其上游激活激酶IKKα。通过敲低OGT降低O-GlcNAcylation可减弱p65的激活磷酸化、核转位、NF-κB转录活性以及NF-κB转录靶标TNF-α和NO的水平；相反，在雨蛙素处理的细胞中，通过PUGNAc提高O-GlcNAc可增加IKKα和p65的O-GlcNAcylation，同时增加p65的磷酸化、活性以及TNF-α和NO的水平。
我们的结果首次证明，OGT介导的O-GlcNAcylation促进胰腺腺泡细胞中的NF-κB信号激活和炎症，这可能促进AP的进展。
