Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago, USA.
Inflamm Bowel Dis. 2011 Mar;17(3):720-31. doi: 10.1002/ibd.21419. Epub 2010 Aug 18.
High levels of proinflammatory cytokines are linked to pathogenesis of diarrhea in inflammatory bowel disease (IBD). Na(+) absorption is compromised in IBD. The studies were designed to determine the effect of tumor necrosis factor-α (TNF-α) on the expression and activity of NHE2, a Na(+) /H(+) exchanger (NHE) that is involved in transepithelial Na(+) absorption in intestinal epithelial cells.
NHE2 regulation was examined in TNF-α-treated C2BBe1 cells by reverse-transcription polymerase chain reaction (RT-PCR), reporter gene assays, and Western blot analysis. NHE isoform activities were measured as ethyl-isopropyl-amiloride- and HOE694-sensitive (22) Na-uptake. In vitro and in vivo protein-DNA interactions were assessed by gel mobility shift assays and chromatin immunoprecipitation studies.
TNF-α treatment of C2BBe1 cells led to repression of NHE2 promoter activity, mRNA, and protein levels; and inhibited both NHE2 and NHE3 mediated (22) Na-uptake. 5'-deletion analysis of the NHE2 promoter-reporter constructs identified basepair -621 to -471 as the TNF-α-responsive region (TNF-RE). TNF-α activated NF-κB subunits, p50 and p65, and their DNA-binding to a putative NF-κB motif within TNF-RE. Mutations in the NF-κB motif abolished NF-κB-DNA interactions and abrogated TNF-α-induced repression. Ectopic overexpression of NF-κB resulted in repression of NHE2 expression. Two functionally distinct inhibitors of NF-κB blocked the inhibitory effect of TNF-α.
The human NHE2 isoform is a direct target of transcription factor NF-κB. TNF-α-mediated activation of NF-κB decreases the expression and activity of NHE2 in the intestinal epithelial cell line, C2BBe1. These findings implicate NF-κB in the modulation of Na(+) absorption during intestinal inflammatory conditions such as IBD where a high level of TNF-α is detected.
高水平的促炎细胞因子与炎症性肠病（IBD）的腹泻发病机制有关。IBD 患者的钠离子吸收受损。本研究旨在探讨肿瘤坏死因子-α（TNF-α）对参与肠道上皮细胞跨上皮钠离子吸收的钠离子/氢（Na + / H + ）交换器（NHE）NHE2 表达和活性的影响。
采用逆转录聚合酶链反应（RT-PCR）、报告基因检测和 Western blot 分析检测 TNF-α 处理的 C2BBe1 细胞中 NHE2 的调节情况。通过乙基-异丙基-amiloride 和 HOE694 敏感（22）Na 摄取来测量 NHE 同工型活性。通过凝胶迁移率变动分析和染色质免疫沉淀研究评估体外和体内的蛋白-DNA 相互作用。
C2BBe1 细胞中 TNF-α 的处理导致 NHE2 启动子活性、mRNA 和蛋白水平的下调，同时抑制了 NHE2 和 NHE3 介导的（22）Na 摄取。NHE2 启动子-报告基因构建体的 5'-缺失分析确定了碱基对-621 至-471 作为 TNF-α 反应区（TNF-RE）。TNF-α 激活了 NF-κB 亚基 p50 和 p65，及其与 TNF-RE 中推定的 NF-κB 基序的 DNA 结合。NF-κB 基序中的突变消除了 NF-κB-DNA 相互作用，并消除了 TNF-α 诱导的抑制作用。NF-κB 的异位过表达导致 NHE2 表达的抑制。NF-κB 的两种功能上不同的抑制剂阻断了 TNF-α 的抑制作用。
人类 NHE2 同工型是转录因子 NF-κB 的直接靶标。TNF-α 介导的 NF-κB 激活降低了肠道上皮细胞系 C2BBe1 中 NHE2 的表达和活性。这些发现表明 NF-κB 参与了肠道炎症条件下（如 IBD）Na + 吸收的调节，在此期间检测到高水平的 TNF-α。
