CEBPD-Mediated Mechanisms of Glucocorticoid Insensitivity in Severe Asthma

项目总结 哮喘是一种间歇性炎症性疾病，影响2500多万美国人，表现为呼吸道疾病。 对特定环境刺激的高反应性。尽管有有效的药物控制哮喘 大多数人，15%的人反应不充分，病情恶化，危及生命。共享的功能 大多数重症患者对糖皮质激素不敏感，这是一种鲜为人知的生理过程。 在哮喘中起重要作用的呼吸道平滑肌(ASM)是糖皮质激素的靶标。 部分通过调节基因转录，改变组蛋白翻译后修饰，以及 转录因子的抑制。CCAAT/增强子结合蛋白D(CEBPD)是一种多效性糖皮质激素。 调节炎症反应、细胞分化和组织的反应转录因子 改建。根据我们的数据显示：(1)致死性哮喘与非哮喘相比，CEBPD的表达较低 ASM，(2)在非哮喘ASM中用糖皮质激素治疗诱导，但在致死性哮喘ASM中不变，以及 (3)通过基因敲除降低CEBPD导致IL-1β诱导的NFkB-荧光素酶表达增加 糖皮质激素刺激，我们的中心假设是，ASM中低水平的CEBPD会引发转录剪裁 以及降低糖皮质激素敏感性的表观遗传修饰。我们将使用 遵循公正和互补的组学方法研究CEBPD对糖皮质激素的影响 致死性哮喘和非哮喘供者的ASM反应：(1)RNA-Seq鉴定转录效应，(2) 蛋白质组学用于确定全局组蛋白翻译后修饰效应，以及(3)芯片序列用于测量 转录因子NFkB与糖皮质激素受体的结合。RNA-Seq的综合分析， 蛋白质组学和芯片序列结果将确定CEBPD影响糖皮质激素反应的主要靶点， 其作用将通过NFkB-荧光素酶检测来确认。我们的项目将提供对转录和 严重哮喘的表观遗传特征使糖皮质激素敏感性生物标志物成为可能 发展，并提供治疗见解，使最脆弱的哮喘患者受益。

PROJECT SUMMARY Asthma is an episodic inflammatory disease that affects over 25 million Americans and manifests as airway hyperresponsiveness to specific environmental stimuli. Despite effective medications that control asthma in most individuals, 15% respond inadequately and suffer life-threatening exacerbations. A feature shared by most patients with severe disease is glucocorticoid insensitivity, a poorly understood physiological process. The airway smooth muscle (ASM), which plays an important role in asthma, is a target of glucocorticoids that act in part via modulation of gene transcription, alteration of histone post-translational modifications, and inhibition of transcription factors. CCAAT/Enhancer Binding Protein D (CEBPD) is a pleiotropic glucocorticoid- responsive transcription factor that regulates inflammatory responses, cell differentiation and tissue remodeling. Based on our data showing that CEBPD expression is (1) lower in fatal asthma vs. non-asthma ASM, (2) induced with glucocorticoid treatment in non-asthma ASM but unchanged in fatal asthma ASM, and that (3) decreasing CEBPD via knockdown resulted in increased IL1β-induced NFkB-luciferase expression with glucocorticoid stimulation, our central hypothesis states that low CEBPD levels in ASM elicits transcriptomic and epigenetic modifications that decrease glucocorticoid sensitivity. We will test this hypothesis by using the following unbiased and complementary `omic approaches to study the effect of CEBPD on glucocorticoid response in ASM from fatal asthma and non-asthma donors: (1) RNA-Seq to identify transcriptomic effects, (2) proteomics to determine global histone post-translational modification effects, and (3) ChIP-Seq to measure transcription factor binding of NFkB and the glucocorticoid receptor. An integrated analysis of RNA-Seq, proteomics and ChIP-Seq results will identify major targets of CEBPD that influence glucocorticoid response, whose role will be confirmed via NFkB-luciferase assays. Our project will offer insights into transcriptional and epigenetic signatures that are characteristic of severe asthma, enable glucocorticoid sensitivity biomarker development, and offer therapeutic insights that benefit the most vulnerable individuals with asthma.