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产生的产生增加有关，这一反应被热休克反应增强。 IL-6是在粘膜中局部产生的，可以调节炎性细胞中肠细胞和IGA中急性相蛋白的合成。此外，IL-6可能会增加严重疾病中的肠道通透性，而从肠道释放的IL-6可能会产生全身作用。尚不清楚粘膜/肠细胞IL-6产生的分子调节以及热休克反应的影响。该项目的目的是检验：1）C/EBP上调IL-6基因的转录，并增加IL-1Beta刺激的Caco-2细胞中IL-6的产生，并且C/EBP通过与包括CBP和P300在内的核Co-Fectors相互作用来发挥其效果； 2）小鼠的败血症和内毒素血症增加体内肠粘膜的C/EBP活性； 3）热休克反应增加了败血症和内毒素小鼠粘膜以及IL-1BETA治疗的CACO-2细胞的C/EBP活性； 4）用蛋白酶体抑制剂治疗CACO-2细胞可诱导热休克反应并激活C/EBP活性和IL-6产生。 CACO-2细胞是人类肠上皮CEIL系，用IL-1Beta处理，C/EBP活性由EMSA确定。进行Supershift分析以确定C/EBP转录因子家族的个体成员的激活。为了确定c/eBP在肠上皮细胞IL-6产生中的作用，用野生型或c/eBP突变的IL-6启动子荧光素酶报道质粒转染了细胞，并用IL-1Beta刺激。在其他实验中，将细胞用C/EBP -beta或-delta的表达质粒转染。通过对C/EBP EMSA进行共免疫沉淀和超速度分析，检查了核共同因素CBP/P300的作用。对于体内实验，败血症是由小鼠皮下注射LPS的盲肠结扎，穿刺和内毒素血症诱导的。 C/EBP活性是在胃肠道（胃，空肠，回肠和结肠）的不同区域的粘膜中确定的。在其他实验中，在诱导败血症或内毒素血症之前，通过高温或注射砷酸钠会诱导热休克反应。在体外实验中，通过用蛋白酶体抑制剂（MG132或乳酸）处理CACO-2细胞也将诱导热休克反应。提出的实验很重要，因为它们将确定败血症和内毒素血症对肠粘膜中C/EBP活性的影响，并确定C/EBP在肠细胞IL-6产生中的作用。该实验还将阐明热休克反应与肠肠球IL-6产生之间的相互作用。由于粘膜IL-6在脓毒症和内毒素血症期间可能具有重要的局部和系统作用，因此对肠道中IL-6的转录调控有了更好的了解，并且使用热冲击反应影响这种调节的可能性将具有重大的临床意义。