Synergistic activation of PTGS2 and CXCL8 by Ni and microbial stimuli

描述(由申请人提供)：拟议项目的总体目标是获得有关颗粒物(PM)空气污染成分和微生物刺激如何协同作用以促进肺部炎症样过程的新信息，从而促进呼吸系统疾病的发生。许多流行病学研究已将接触PM与慢性呼吸系统和心血管疾病联系起来。研究表明，PM的常见成分镍在体外和体内都介导了PM的一些生物学效应。慢性炎症性疾病也与包括发酵支原体在内的感染性病原体的定植有关。炎症的特征之一是通过诱导环氧合酶-2酶(前列腺素-内过氧化物酶合酶2，Ptgs2)增加前列腺素(PG)的合成。初步数据显示，NiS04和发酵分枝杆菌来源的脂肽MALP-2可协同增强Ptgs2和免疫调节趋化因子CXCL8(IL-8)在培养的人肺成纤维细胞(HLF)中的表达。在Ptgs2和CXCL8诱导的同时，Ni和MALP-2协同刺激HLF释放PGE2和CXCL8蛋白。这项拟议的研究将探索这样的假设，即Ptgs2衍生的PGE2有助于在镍和微生物刺激的混合暴露期间从HLF产生CXCL8的放大。其具体目的是1)确定协同诱导Ptgs2在HLF中表达的细胞和分子机制，以响应Ni和MALP-2的同时暴露；2)确定Ni和MALP-2混合暴露后PGE2的产生和随后CXCL8的产生之间的机制关系。由人Ptgs2和CXCL8启动子驱动的荧光素酶报告质粒以及凝胶迁移率改变分析(EMSA)将用于确定Ni和MALP-2如何协同增强Ptgs2和CXCL8的激活。相关的顺式调控DNA结合元件和反式激活蛋白因子将使用全长、截短和突变版本的Ptgs2和CXCL8启动子进行定位。SiRNA将与药理激动剂和拮抗剂一起用于探讨Ptgs2、PGE2和PGE2受体亚型在Ni和MALP-2诱导的CXCL8表达和随后的HLF蛋白释放中所起的特殊作用。现实生活中的环境暴露很可能涉及毒物和微生物刺激的混合物。这项研究的结果将增加我们对镍等金属如何使细胞对微生物驱动的肺部炎症敏感，以及毒物和微生物刺激如何相互作用以促进人类呼吸道疾病发病的理解。

DESCRIPTION (provided by applicant): The overall objective of the proposed project is to gain novel information regarding how components of particulate matter (PM) air pollution and microbial stimuli synergistically interact to promote inflammatory-like processes in the lung, and thereby contribute to the onset of respiratory disease. Numerous epidemiological studies have linked exposure to PM with chronic respiratory and cardiovascular diseases. Studies have shown nickel, a common component of PM, mediates some of the biological effects of PM both in vitro and in vivo. Chronic inflammatory diseases are also associated with colonization by infectious agents, including Mycoplasma fermentans. One of the hallmarks of inflammation is increased elaboration of prostaglandins (PG) through the induction of the cyclooxygenase-2 enzyme (prostaglandin-endoperoxidase synthase 2, PTGS2). Preliminary data show that exposure to NiS04 and the M. fermentans-derived lipopeptide MALP-2 synergistically enhances expression of PTGS2 and the immune-modulating chemokine CXCL8(IL-8) in cultured human lung fibroblast cells (HLF). Concurrent with the induction of PTGS2 and CXCL8, Ni and MALP-2 synergistically stimulate PGE2 and CXCL8 protein release from HLF. The proposed study will explore the hypothesis that PTGS2-derived PGE2 contributes to the amplification of CXCL8 production from HLF during mixed exposures to Ni and microbial stimuli. The specfic aims are to 1) determine the cellular and molecular mechanisms underlying the synergistic induction of PTGS2 expression in HLF in response to concurrent Ni and MALP-2 exposure and 2) determine the mechanistic relationship between the production of PGE2 following mixed exposures to Ni and MALP-2 and subsequent production of CXCL8. Luciferase reporter plasmids driven by human PTGS2 and CXCL8 promoters along with electrophoretic mobility shift assays (EMSA) will be employed to determine how Ni and MALP-2 synergistically enhance activation of PTGS2 and CXCL8. Relevant cis-regulatory DNA binding elements and transactivating protein factors responsible will be mapped using full-length, truncated and mutant versions of the PTGS2 and CXCL8 promoters. siRNA along with pharmacological agonists and antagonists will be used to probe the specific contribution of PTGS2, PGE2 and PGE2 receptor subtypes involved in transducing Ni and MALP-2-induced CXCL8 expression and subsequent protein release from HLF. Real-life environmental exposures are likely to involve a mixture of toxicants and microbial stimuli. Results from this study will increase our understanding of how metals such as Ni can sensitize cells to microbial-driven inflammation in the lung, and how toxicant and microbial stimuli interact to promote the onset of respiratory disease in human populations.