C/EBP and IL-6 Production in Mucosa and Enterocytes

粘膜和肠上皮细胞中 C/EBP 和 IL-6 的产生

• 批准号: 6743728
• 负责人: PER-OLOF J HASSELGREN
• 金额: $ 29.64万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6743728
• 关键词: binding proteins; biological signal transduction; cofactor; gene expression; genetic transcription; immunoprecipitation; interleukin 6; intestinal mucosa; laboratory mouse; northern blottings; nuclear factor kappa beta; protein structure function; septicemia; site directed mutagenesis; tissue /cell culture; transcription factor; transfection; western blottings

DESCRIPTION (provided by applicant): Sepsis and endotoxemia are associated with increased production of IL-6 in intestinal mucosa and enterocytes, a response that is augmented by the heat shock response. IL-6, locally produced in the mucosa, may regulate the synthesis of acute phase proteins in enterocytes and IgA in inflammatory cells. In addition, IL-6 may increase intestinal permeability in critical illness and IL-6 released from the gut may have systemic effects. The molecular regulation of mucosal/ enterocyte IL-6 production, as well as the influence of the heat shock response, are not well understood. The purpose of this project is to test the hypotheses that: 1) C/EBP upregulates transcription of the IL-6 gene and increases IL-6 production in IL-1beta-stimulated Caco-2 cells and that C/EBP exerts its effect by interacting with nuclear co-factors, including CBP and p300; 2) sepsis and endotoxemia in mice increase C/EBP activity in intestinal mucosa in vivo; 3) the heat shock response increases C/EBP activity in mucosa of septic and endotoxemic mice and IL-1beta-treated Caco-2 cells; 4) treatment of Caco-2 cells with proteasome inhibitors induces the heat shock response and activates C/EBP activity and IL-6 production. Caco-2 cells, a human intestinal epithelial ceil line, are treated with IL-1beta and C/EBP activity is determined by EMSA. Supershift analysis is performed to determine the activation of individual members of the C/EBP family of transcription factors. To determine the role of C/EBP in enterocyte IL-6 production, cells are transfected with wild-type or C/EBP mutated IL-6 promoter luciferase reporter plasmid and stimulated with IL-1beta. In other experiments, cells are transfected with expression plasmids for C/EBP-beta or -delta. The role of the nuclear co-factors CBP/p300 is examined by performing co-immunoprecipitation and supershift analysis of C/EBP EMSAs. For in vivo experiments, sepsis is induced by cecal ligation and puncture and endotoxemia by subcutaneous injection of LPS in mice. C/EBP activity is determined in mucosa from different regions of the gastrointestinal tract (stomach, jejunum, ileum and colon). In other experiments, the heat shock response is induced in mice by hyperthermia or injection of sodium arsenite before induction of sepsis or endotoxemia. In the in vitro experiments, heat shock response will also be induced by treating Caco-2 cells with proteasome inhibitors (MG132 or lactacystin). The proposed experiments are important because they will determine the influence of sepsis and endotoxemia on C/EBP activity in intestinal mucosa and define the role of C/EBP in enterocyte IL-6 production. The experiments will also elucidate the interaction between heat shock response and enterocyte IL-6 production. Because mucosal IL-6 may have important local as well as systemic effects during sepsis and endotoxemia, a better understanding of the transcriptional regulation of IL-6 in the intestine and the possibility of influencing this regulation with the heat shock response will have significant clinical implications.

描述(由申请人提供)： 败血症和内毒素血症与肠粘膜和肠细胞中IL-6的产生增加有关，这种反应因热休克反应而增强。IL-6在粘膜局部产生，可能调节肠细胞合成急性期蛋白，调节炎症细胞合成IgA。此外，在危重疾病中，IL-6可能会增加肠道通透性，从肠道释放的IL-6可能具有全身效应。粘膜/肠细胞产生IL-6的分子调控，以及热休克反应的影响，还不是很清楚。1)C/EBP上调IL-1β刺激的Caco-2细胞中IL-6基因的转录，增加IL-6的产生，C/EBP通过与CBP、p300等核共因子相互作用发挥作用；2)小鼠脓毒症和内毒素血症增加活体肠粘膜C/EBP活性；3)热休克反应增加败血症和内毒素血症小鼠及经IL-1β处理的Caco-2细胞的C/EBP活性；4)蛋白酶体抑制剂处理Caco-2细胞可诱导热休克反应，激活C/EBP活性和IL-6的产生。人肠上皮细胞系Caco-2细胞经IL-1β处理后，用EMSA法测定C/EBP活性。超漂移分析用于确定C/EBP转录因子家族的单个成员的激活。为了确定C/EBP在肠上皮细胞产生IL-6中的作用，将野生型或C/EBP突变的IL-6启动子荧光素酶报告基因导入细胞，并用IL-1β刺激。在其他实验中，将C/EBP-β或-Delta的表达载体导入细胞。通过对C/EBP EMSA进行免疫共沉淀和超位移分析，研究了核辅助因子CBP/p300的作用。在体内实验中，通过盲肠结扎和穿孔造成脓毒症，通过皮下注射内毒素引起小鼠内毒素血症。C/EBP活性测定来自胃肠道不同区域(胃、空肠、回肠和结肠)的粘膜。在其他实验中，在诱导败血症或内毒素血症之前，通过高温或注射亚砷酸钠来诱导小鼠的热休克反应。在体外实验中，用蛋白酶体抑制剂(MG132或lactacystin)处理Caco-2细胞也可以诱导热休克反应。这些实验很重要，因为它们将确定败血症和内毒素血症对肠粘膜C/EBP活性的影响，并确定C/EBP在肠细胞产生IL-6中的作用。这些实验还将阐明热休克反应和肠道细胞产生IL-6之间的相互作用。由于IL-6在脓毒症和内毒素血症中可能具有重要的局部和全身效应，因此更好地了解IL-6在肠道中的转录调控以及通过热休克反应影响这一调控的可能性将具有重要的临床意义。