Kinetics of lipopolysaccharide-induced transcription factor activation/inactivation and relation to proinflammatory gene expression in the murine spleen.
Kinetics of lipopolysaccharide-induced transcription factor activation/inactivation and relation to proinflammatory gene expression in the murine spleen.
复制标题
脂多糖诱导的转录因子激活/失活的动力学及其与小鼠脾脏中促炎基因表达的关系。
DOI：
10.1016/s0041-008x(02)00077-7
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发表时间：
2003
影响因子：
3.8
通讯作者：
Pestka,JamesJ
中科院分区：
文献类型：
--
作者：
Zhou,Hui-Ren;Islam,Zahidul;Pestka,JamesJ
细菌脂多糖（LPS）通过诱导促炎细胞因子基因表达来诱发炎症和内毒素休克。本研究的目的是测试的假设，即差异激活的转录因子结合在脾脏中与促炎细胞因子基因表达在小鼠暴露于LPS。在8 h内对腹腔注射4 mg/kg LPS的小鼠脾脏中的促炎细胞因子表达进行评价时，肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1β和IL-6 mRNA分别升高至溶剂对照组的5倍、6倍和300倍。TNF- α和IL-6 mRNA均在2 h达到峰值，此后开始下降，而IL-1β mRNA在2 h至8 h保持升高。脾核蛋白结合与促炎细胞因子启动子相关的六种不同共有转录控制基序的能力也在8小时内测量。电泳迁移率变动分析（EMSA）显示，活化蛋白-1（AP-1）的结合活性在0.5 ~ 8 h显著增加，CCAAT增强子结合蛋白（C/EBP）和核因子κB（NF-κB）的结合活性在0.5 ~ 1.5 h也显著增加。在0.5小时，环磷酸腺苷反应元件（CRE）结合蛋白（CREB）和结合略有升高，而激活蛋白-2（AP-2）和特异性蛋白1（Sp1）结合不受影响。抗体超位移EMSA和蛋白质印迹分析证实，这些因子的结合增加与LPS诱导的AP-1（c-Jun、磷酸化c-Jun、Jun D和Jun B）、C/EBPβ、NF-κB（p50、p65和c-Rel）、CREB（CREB-1、CREB-2和ATF-2）和AP-2α蛋白的核浓度增加相关。值得注意的是，8小时后，C/EBP，CREB，AP-2，和Sp1结合活性大大耗尽相对于幼稚和相应的媒介物对照。当小鼠暴露于第二剂量的LPS时，在4 mg/kg引发剂量后8h，TNF-α和IL-6 mRNA反应显著受损，表明小鼠在该时间点具有内毒素耐受性。两者合计，在该模型中观察到的转录因子结合的静止、活跃和抑制阶段与体内LPS诱导的促炎细胞因子表达的快速瞬时性质以及对二次LPS暴露的耐受性高度一致。
Bacterial lipopolysaccharide (LPS) elicits inflammation and endotoxic shock by inducing proinflammatory cytokine gene expression. The purpose of this study was to test the hypothesis that differential activation of transcription factor binding in the spleen correlates with proinflammatory cytokine gene expression in mice exposed to LPS. When proinflammatory cytokine expression in spleen was evaluated in mice injected ip with 4 mg/kg LPS over an 8-h period, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 mRNAs were elevated up to 5-, 6-, and 300-fold, respectively, over vehicle controls. Both TNF- α and IL-6 mRNA peaked at 2 h and begin to decline thereafter, whereas IL-1β mRNA remained elevated from 2 to 8 h. The capacities of splenic nuclear proteins to bind to six different consensus transcriptional control motifs associated with proinflammatory cytokine promoters were also measured over 8 h. Electrophoretic mobility shift assay (EMSA) revealed that binding activity was markedly increased at 0.5 to 8 h for activator protein-1 (AP-1) as were CCAAT enhancer-binding protein (C/EBP) and nuclear factor κB (NF-κB) at 0.5 to 1.5 h. At 0.5 h, cyclic AMP response element (CRE)-binding protein (CREB) and binding was slightly elevated, whereas activator protein- 2 (AP-2) and specificity protein 1 (Sp1) binding were not affected. Antibody supershift EMSA and Western blot analysis confirmed that increased binding of these factors correlated with LPS-induced increases in nuclear concentrations of AP-1 (c-Jun, phosphorylated c-Jun, Jun D, and Jun B), C/EBPβ, NF-κB (p50, p65, and c-Rel), CREB (CREB-1, CREB-2, and ATF-2), and AP-2α proteins. Remarkably, after 8 h, C/EBP, CREB, AP-2, and Sp1 binding activities were greatly depleted relative to both naive and corresponding vehicle controls. When mice were exposed to a second dose of LPS, 8 h after a 4 mg/kg priming dose, TNF-α and IL-6 mRNA responses were markedly impaired, suggesting that the mice were endotoxin tolerant at this time point. Taken together, the quiescent, active, and suppressive phases of transcription factor binding observed in this model were highly consistent with the rapid transient nature of LPS-induced proinflammatory cytokine expression in vivo as well as tolerance to secondary LPS exposure.