Synergistic activation of PTGS2 and CXCL8 by Ni and microbial stimuli

Ni和微生物刺激对PTGS2和CXCL8的协同激活

• 批准号: 7483627
• 负责人: Kelly Brant
• 金额: $ 5.13万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7483627
• 关键词: Acute respiratory infection; Adenylate Cyclase; Agonist; Air Pollution; Aromatic Polycyclic Hydrocarbons; Attention; Binding Sites; Biological; Cardiovascular Diseases; Cells; Chemicals; Chronic; Condition; Cyclic AMP; DNA Binding; Data; Development; Dinoprostone; Disease; EP4 receptor; Electrophoretic Mobility Shift Assay; Elements; Environmental Exposure; Environmental Pollutants; Enzymes; Epidemiologic Studies; Exposure to; Fibroblasts; Gene Expression; Gene Proteins; Genes; Human; IL8 gene; Immune; In Vitro; Infectious Agent; Inflammation; Inflammatory; Inflammatory Response; Length; Life; Link; Luciferases; Lung; Lung diseases; Maps; Measures; Mediating; Messenger RNA; Metals; Molecular; Mycoplasma fermentans; NS-398; Natural Immunity; Nickel; PTGS2 gene; Particulate Matter; Pathway interactions; Pattern; Plasmids; Population; Process; Production; Property; Prostaglandin E Receptor; Prostaglandin Inhibition; Prostaglandins; Proteins; Relative (related person); Reporter; Research; Role; Small Interfering RNA; Source; Stimulus; Stress; Testing; Toll-Like Receptor 2; Toll-like receptors; Toxic effect; Transcriptional Activation; Western Blotting; chemokine; cyclooxygenase 2; cytokine; design; in vivo; inhibitor/antagonist; mRNA Expression; macrophage stimulatory lipopeptide 2; microbial; mutant; novel; promoter; protein expression; respiratory; response; toxicant; transcription factor

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.

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）空气污染和微生物刺激成分如何协同相互作用以促进肺部炎症样过程的新信息，从而促进呼吸道疾病的发作。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，镍是PM的常见成分，在体内和体外介导PM的一些生物学效应。慢性炎症性疾病也与感染因子的定植有关，包括发酵支原体。炎症的标志之一是通过诱导环加氧酶-2（前列腺素内过氧化物酶合成酶2，PTGS2）增加前列腺素（PG）的精化。初步数据显示，暴露于NiS04和M. fermentens衍生的脂肽MALP-2可协同增强培养的人肺成纤维细胞（HLF）中PTGS2和免疫调节趋化因子CXCL8（IL-8）的表达。在诱导PTGS2和CXCL8的同时，Ni和MALP-2协同刺激PGE2和CXCL8蛋白从HLF中释放。该研究将探索ptgs2衍生的PGE2在混合暴露于Ni和微生物刺激时促进HLF中CXCL8生成的假设。具体目的是：1)确定Ni和MALP-2同时暴露在HLF中协同诱导PTGS2表达的细胞和分子机制；2)确定Ni和MALP-2混合暴露后PGE2的产生与随后CXCL8的产生之间的机制关系。由人类PTGS2和CXCL8启动子驱动的荧光素酶报告质粒以及电泳迁移率转移试验（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结合元件和相关的反激活蛋白因子将使用PTGS2和CXCL8启动子的全长、截短和突变版本进行定位。siRNA将与药理学激动剂和拮抗剂一起用于探测PTGS2、PGE2和PGE2受体亚型参与Ni和malp -2诱导的CXCL8表达和随后的HLF蛋白释放的特异性贡献。现实生活中的环境暴露可能涉及有毒物质和微生物刺激的混合物。这项研究的结果将增加我们对金属（如Ni）如何使细胞对肺部微生物驱动的炎症敏感，以及有毒物质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。