Regulation of Surfactant Synthesis in Asthma

哮喘中表面活性剂合成的调节

• 批准号: 7214736
• 负责人: Rama K Mallampalli
• 金额: $ 31.47万
• 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 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7214736
• 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，一种高度富含磷脂酰胆碱（PC）的材料，缺乏哮喘。肿瘤坏死因子α（TNFA）在哮喘中起关键作用，并降低表面活性剂PC合成。该提案中解决的主要问题是，TNFA如何减少气道炎症期间的PC合成？ PI实验室中的先前研究表明，生物活性鞘脂，鞘氨醇是响应TNFA激活的鞘氨素水解途径而产生的，它是TNFA对表面活性剂PC合成的抑制作用的一种重要机制。该建议将通过研究分子基础来扩展这些观察结果，而TNFA-弹性信号传导抑制表面活性剂PC的合成。细胞中PC的合成受速率限制酶胞化酶（CT）的严格调节。 CT活性受到鞘氨酸和酶磷酸化的抑制。然而，CT磷酸化和CT基因转录调节的生理作用仍然很大未知。 TNFA的一种影响是多个激酶途径的激活，包括C-Jun N末端激酶（JNK）途径。 PI的初步数据表明，i）鞘氨醇和JNK水平升高，但在气道炎症的鼠卵形蛋白模型中CT表达降低，II）鞘氨氨酸会协同激活JNK，增加CT磷酸化，并降低CT mRNA。因此，鞘氨酸导致CT磷酸化或CT基因转录抑制的JNK的TNFA激活可能代表了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型效应机制。我们将特异性检验以下假设：TNFA通过协同增加CT磷酸化（AIM 1）并降低CT基因转录（AIM 2）来抑制表面活性剂PC的合成（AIM 2），从而在过敏性炎症的鼠模型中降低了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的鞘氨醇激活引起的（AIM 1）。我们还将确定鞘氨酸是否抑制CT基因转录（AIM 2）。在AIM 1中，我们的假设将使用分子和生化方法进行检验，以鉴定CT主要结构内的区域，这些区域是JNK对位点特异性磷酸化的靶标。我们将执行CT的位置定向诱变，以产生酶突变体，该酶突变体在体内表达时对JNK诱导的磷酸化的敏感性不太敏感。在AIM 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C/S-S-SiS-SIS-SICTING负调控元件（S）（NRE）。最后，包括这些元素的CT基因的5'侧翼区域的一部分与报告基因（B-半乳糖苷酶）耦合，并用于产生转基因启动子重生子小鼠。我们将检验以下假设：这些元素足以在过敏（TH2）炎症模型中赋予CT基因转录的TNFA抑制。我们的假设将使用气道炎症的椭圆蛋白鼠模型进行检验，并在原发性II型肺泡上皮细胞中进行分析。这些研究将补充TNFA响应性的II型细胞系。该提案影响哮喘领域的独特贡献包括1）描述一种新型激酶途径，将TNFA信号与表面活性剂合成联系起来，2）研究研究了CT基因转录抑制作用，该研究代表了该关键表面活性剂酶和这种相对新的调节机制，该机制相对较新3）首次通过表达具有强大的催化活性的新型CT突变体的表达来刺激表面活性剂合成的研究，并在与细胞因子相关的气道炎症的情况下具有抗磷酸化。