Mejías-Luque Raquel, Peiró Sandra, Vincent Audrey, Van Seuningen Isabelle, de Bolós Carme
IMIM-Hospital del Mar (PRBB), Dr. Aiguader, 88, 08003 Barcelona, Spain.
Biochim Biophys Acta. 2008 Oct;1783(10):1728-36. doi: 10.1016/j.bbamcr.2008.05.020. Epub 2008 Jun 4.
The gastric mucosal levels of the pro-inflammatory cytokine Interleukin 6 (IL-6) have been reported to be increased in Helicobacter pylori-infected subjects and, in gastric adenocarcinomas, the up-regulation of intestinal mucin genes (MUC2 and MUC4) has been detected. To analyse the regulatory effects of IL-6 on the activation of intestinal mucins, six gastric cancer cell lines were treated for different times with several concentrations of IL-6, and the expression of MUC2 and MUC4 was evaluated. IL-6 induced MUC4 expression, detected by quantitative RT-PCR, Western blot and immunofluorescence, and MUC2 expression was not affected. MUC4 mRNA levels decreased after blocking the gp130/STAT3 pathway at the level of the receptor, and at the level of STAT3 activation using the AG490 specific inhibitor. MUC4 presents two putative binding sites for STAT factors that may regulate MUC4 transcription after a pro-inflammatory stimulus as IL-6. By EMSA, ChIP and site-directed mutagenesis we show that STAT3 binds to a cis-element at -123/-115, that conveys IL-6 mediated up-regulation of MUC4 transcriptional activity. We also demonstrated that p-STAT3 binds to MUC4 promoter and a three-fold increase in p-STAT3 binding was observed after treating GP220 cells with IL-6. In conclusion, IL-6 treatment induced MUC4 expression through the gp130/STAT3 pathway, indicating the direct role of IL-6 on the activation of the intestinal mucin gene MUC4 in gastric cancer cells.
据报道，幽门螺杆菌感染患者胃黏膜中促炎细胞因子白细胞介素6（IL-6）水平升高，并且在胃腺癌中已检测到肠黏液素基因（MUC2和MUC4）上调。为了分析IL-6对肠黏液素激活的调节作用，用几种浓度的IL-6对六种胃癌细胞系进行不同时间的处理，并评估MUC2和MUC4的表达。通过定量RT-PCR、蛋白质免疫印迹和免疫荧光检测发现，IL-6诱导了MUC4的表达，而MUC2的表达未受影响。在受体水平阻断gp130/STAT3信号通路以及使用AG490特异性抑制剂在STAT3激活水平阻断后，MUC4 mRNA水平降低。MUC4存在两个可能的STAT因子结合位点，在受到如IL-6这样的促炎刺激后，可能调节MUC4转录。通过电泳迁移率变动分析（EMSA）、染色质免疫沉淀（ChIP）和定点诱变，我们发现STAT3与-123/-115处的顺式元件结合，该元件介导IL-6介导的MUC4转录活性上调。我们还证明p-STAT3与MUC4启动子结合，在用IL-6处理GP220细胞后，观察到p-STAT3结合增加了三倍。总之，IL-6处理通过gp130/STAT3信号通路诱导MUC4表达，表明IL-6在胃癌细胞中对肠黏液素基因MUC4激活具有直接作用。
