Regulation of Surfactant Synthesis in Asthma

哮喘中表面活性剂合成的调节

• 批准号: 7386769
• 负责人: Rama K Mallampalli
• 金额: $ 16.36万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7386769
• 关键词: 5' Flanking Region; Address; Agonist; Allergic; Alveolar; Alveolus; Asthma; Be++ element; Beryllium; Biochemical; Cartoons; Cell Line; Cells; Ceramidase; Choline-Phosphate Cytidylyltransferase; Class; Complex Mixtures; Coupled; Cytidylyltransferase; Data; Disease; Elements; Enzyme Inhibition; Enzymes; Epithelial Cells; Epithelium; Exhibits; Fatty Acids; Foundations; Galactosidase; Generations; Genes; Genetic Transcription; Goals; Hydrolysis; In Vitro; Inflammation; Inflammatory; Investigation; Laboratories; Lead; Lecithin; Left; Link; Lipids; Localized; Lung; Lung Inflammation; Mediator of activation protein; Messenger RNA; Modeling; Molecular; Mus; Mutagenesis; Obstruction; Ovalbumin; Pathway interactions; Phosphorylation; Phosphotransferases; Physiological; Pilot Projects; Play; Prevalence; Production; Proline; Protein-Serine-Threonine Kinases; Proteins; Rate; Regulation; Regulatory Element; Replacement Therapy; Reporter; Reporter Genes; Repression; Research Personnel; Resistance; Role; Second Messenger Systems; Severities; Signal Transduction; Site; Site-Directed Mutagenesis; Sphingolipids; Sphingomyelinase; Sphingomyelins; Sphingosine; Staphylococcus aureus glutamic acid-specific endopeptidase; Structure; Surface Tension; System; Testing; Time Study; Transgenic Organisms; Tumor Necrosis Factor-alpha; Type II Epithelial Receptor Cell; airway inflammation; airway obstruction; airway remodeling; allergic airway inflammation; base; cytokine; enzyme activity; gene repression; in vivo; lipid biosynthesis; mutant; novel; programs; promoter; response; second messenger; stress-activated protein kinase 1; surfactant; surfactant deficiency

DESCRIPTION (provided by applicant): Surfactant, a material highly enriched with phosphatidylcholine (PC), is deficient in asthma. Tumor necrosis factor alpha (TNFa) plays a key role in asthma and decreases surfactant PC synthesis. The major question addressed in this proposal is how TNFa decreases PC synthesis during airway inflammation? Prior studies in the PI's laboratory have shown that the bioactive sphingolipid, sphingosine generated in response to TNFa activation of the sphingomyelin hydrolysis pathway, is one important mechanism whereby TNFa exerts its inhibitory effects on surfactant PC synthesis. This proposal will expand on these observations by investigating the molecular basis by which TNFa-sphingosine signaling inhibits surfactant PC synthesis. The synthesis of PC in cells is tightly regulated by the rate-limiting enzyme cytidylyltransferase (CT). CT activity is inhibited by sphingosine and by enzyme phosphorylation. The physiologic role of CT phosphorylation and regulation of CT gene transcription, however, remain largely unknown. One effect of TNFa is the activation of multiple kinase pathways, including the c-Jun N-terminal kinase (JNK) pathway. Preliminary data by the PI demonstrate that i) sphingosine and JNK levels are elevated but CT expression decreased in a murine ovalbumin model of airway inflammation, and ii) sphingosine coordinately activates JNK, increases CT phosphorylation, and decreases CT mRNA. Thus, TNFa activation of JNK leading to CT phosphorylation or CT gene transcriptional repression by sphingosine might represent a novel effector mechanism for the inhibitory effects of TNFa on surfactant PC synthesis in allergic airway inflammation. We will specifically test the hypothesis that TNFa inhibits surfactant PC synthesis by coordinately increasing CT phosphorylation (AIM 1) and decreasing CT gene transcription (AIM 2) thereby reducing surfactant availability in a murine model of allergic inflammation. We will determine if negative effects of TNFa on CT activity are due to sphingosine activation of JNK (AIM 1). We will also determine if sphingosine inhibits CT gene transcription (AIM 2). In Aim 1 our hypothesis will be tested using molecular and biochemical approaches to identify the regions within the CT primary structure that are targets for site-specific phosphorylation by JNK. We will perform site-directed mutagenesis of CT to generate enzyme mutants, that when expressed in vivo, are less sensitive to JNK-induced phosphorylation. In Aim 2, we will perform deletional and mutational analysis of the CT promoter to localize sphingosine-regulated c/s-acting negative regulatory element(s) (NRE). Finally, a portion of the 5' flanking region of the CT gene including these elements was coupled to a reporter gene (b-galactosidase) and used to generate transgenic promoter-reporter mice. We will test the hypothesis that these elements are sufficient to confer TNFa-repression of CT gene transcription within a model of allergic (Th2) inflammation. Our hypothesis will be tested using an ovalbumin murine model of airway inflammation with analysis conducted in primary type II alveolar epithelial cells. These studies will be supplemented with a TNFa-responsive murine type II cell line. The unique contributions of this proposal impacting the field of asthma include 1) delineation of a novel kinase pathway linking TNFa-signaling with surfactant synthesis, 2) studies investigating CT gene transcriptional repression which represent a relatively new regulatory mechanism for this key surfactant enzyme, and 3) for the first time, studies directed at stimulating surfactant synthesis by expression of novel CT mutants that exhibit robust catalytic activity and are phosphorylation-resistant in the setting of cytokine-associated airway inflammation.

描述（由申请人提供）： 表面活性剂是一种富含磷脂酰胆碱（PC）的物质，在哮喘中是缺乏的。肿瘤坏死因子 α (TNFa) 在哮喘中起关键作用并减少表面活性​​剂 PC 的合成。该提案解决的主要问题是 TNFa 如何在气道炎症期间减少 PC 合成？ PI实验室先前的研究表明，TNFa激活鞘磷脂水解途径时产生的生物活性鞘脂（鞘氨醇）是TNFa对表面活性剂PC合成发挥抑制作用的重要机制之一。该提案将通过研究 TNFa-鞘氨醇信号传导抑制表面活性剂 PC 合成的分子基础来扩展这些观察结果。细胞内 PC 的合成受到限速酶胞苷酰转移酶 (CT) 的严格调控。 CT 活性受到鞘氨醇和酶磷酸化的抑制。然而，CT 磷酸化的生理作用和 CT 基因转录的调节仍然很大程度上未知。 TNFa 的作用之一是激活多种激酶途径，包括 c-Jun N 末端激酶 (JNK) 途径。 PI 的初步数据表明，i) 在气道炎症的小鼠卵清蛋白模型中，鞘氨醇和 JNK 水平升高，但 CT 表达降低；ii) 鞘氨醇协调激活 JNK，增加 CT 磷酸化，并减少 CT mRNA。因此，TNFa 激活 JNK 导致 CT 磷酸化或鞘氨醇 CT 基因转录抑制可能代表了 TNFa 对过敏性气道炎症中表面活性剂 PC 合成的抑制作用的新效应机制。我们将具体测试以下假设：TNFa 通过协调增加 CT 磷酸化 (AIM 1) 和减少 CT 基因转录 (AIM 2) 来抑制表面活性剂 PC 合成，从而减少过敏性炎症小鼠模型中表面活性剂的可用性。我们将确定 TNFa 对 CT 活性的负面影响是否是由于 JNK (AIM 1) 的鞘氨醇激活所致。我们还将确定鞘氨醇是否抑制 CT 基因转录 (AIM 2)。在目标 1 中，我们的假设将使用分子和生化方法进行测试，以确定 CT 一级结构内的区域，这些区域是 JNK 进行位点特异性磷酸化的目标区域。我们将对 CT 进行定点诱变以产生酶突变体，这些突变体在体内表达时对 JNK 诱导的磷酸化不太敏感。在目标 2 中，我们将对 CT 启动子进行缺失和突变分析，以定位鞘氨醇调节的顺/顺作用负调节元件 (NRE)。最后，将包含这些元件的 CT 基因 5' 侧翼区域的一部分与报告基因（b-半乳糖苷酶）偶联，并用于产生转基因启动子-报告小鼠。我们将测试以下假设：这些元件足以在过敏性 (Th2) 炎症模型中对 CT 基因转录产生 TNFa 抑制。我们的假设将使用卵清蛋白小鼠气道炎症模型进行测试，并在原代 II 型肺泡上皮细胞中进行分析。这些研究将得到 TNFa 反应性小鼠 II 型细胞系的补充。该提案对哮喘领域的独特贡献包括 1) 描绘了一种将 TNFa 信号传导与表面活性剂合成联系起来的新型激酶途径，2) 研究 CT 基因转录抑制，这代表了这种关键表面活性剂酶的相对较新的调节机制，以及3) 首次通过表达新型 CT 突变体来刺激表面活性剂合成的研究，这些突变体在细胞因子相关气道炎症中表现出强大的催化活性并且具有磷酸化抗性。