Mechanisims Regulation Functions of Airway Smooth Muscle

气道平滑肌的调节功能机制

• 批准号: 7098904
• 负责人: YASSINE AMRANI
• 金额: $ 34.82万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-05 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7098904
• 关键词: CD38 molecule; asthma; autocrine; biological signal transduction; cell adhesion molecules; chemokine; clinical research; cytokine; flow cytometry; gel mobility shift assay; gene expression; genetic transcription; genetically modified animals; growth factor; human tissue; immunoprecipitation; inflammation; interferon beta; laboratory mouse; nuclear factor kappa beta; paracrine; respiratory muscles; smooth muscle; transcription factor; tumor necrosis factor alpha

DESCRIPTION (provided by applicant): The mechanisms that orchestrate and/or perpetuate chronic airway inflammation, believed to be key factors in the progression of asthma, remain unknown. Recent evidence suggests that airway smooth muscle (ASM) synthetic function, defined as secretion of cytokines, chemokines or growth factors and expression of adhesion molecules, may play an active role in regulating airway inflammation in asthma. In the previous funding period, we identified multiple transcription factors such as NF-kappaB and NF-AT that regulate cytokine-induced inflammatory gene expression in human ASM cells. In our new preliminary studies, we found that TNFalpha activates other transcription factors IRF-1, STAT1 and STAT2, and differentially regulates the expression of CD38, IL-6, RANTES and eotaxin but not ICAM-1 via the autocrine action of secreted IFNbeta. Accordingly, the aims of this COMPETING CONTINUATION proposal seek to extend our previous findings by defining the relative contribution of each of these signaling molecules in the regulation of TNFalpha effects on synthetic functions. The central hypothesis of this proposal states that TNFalpha and autocrine IFNbeta act in synergy to differentially regulate inflammatory gene expression via the coordinated activation of STAT1/STAT2, NF-kappaB and CD38-dependent pathways. In Aim 1, we will determine whether NF-kappaB functionally interacts with STAT1/STAT2 and IRF-1 to regulate TNFalpha-induced expression of inflammatory genes in an IFNbeta-dependent manner using cytokine promoter constructs and coimmunoprecipitation, co-localization and gel shift assays. In Aim 2, we will identify the transcriptional mechanisms (TRAFIKK) regulating cytokine-induced IFNbeta expression in human ASM cells using dominant negative and constitutively active cDNA constructs. We will also determine the in vivo relevance of IFNbeta expression by ASM in a murine model of bronchial hyper-responsiveness by characterizing the time course of in vivo expression and/or activation of IFNbeta signaling molecules in ASM. In Aim 3, we will characterize whether IFNbeta-dependent CD38 pathways are necessary and/or sufficient to regulate TNFalpha-induced expression of inflammatory genes in ASM cells. The role of CD38 will be investigated using CD38 -/- ASM and soluble inhibitors. The role of calcium-dependent pathways in TNFalpha-inducible genes will be assessed by examining the role of FKBP12.6 and NF-AT using gene-deficient mice, reporter constructs and soluble inhibitors. Because of the importance of inflammatory cytokines/chemokines in asthma, results from our studies will determine that ASM-derived cytokines participate in the regulation of airway inflammation not only via paracrine effects but also via the autocrine modulation of ASM synthetic function. Thus, understanding the transcriptional mechanisms regulating the expression of inflammatory genes in ASM will likely lead to new therapeutic approaches for the treatment of asthma.

描述（由申请人提供）：编排和/或永存的慢性气道炎症的机制，据信是哮喘进展的关键因素，仍然未知。最近的证据表明，气道平滑肌（ASM）合成功能（定义为细胞因子，趋化因子或生长因子的分泌以及粘附分子的表达）可能在调节哮喘中气道炎症中起积极作用。在上一个资金期间，我们确定了多个转录因子，例如NF-kappab和NF-AT，它们调节了人ASM细胞中细胞因子诱导的炎症基因表达。在我们的新初步研究中，我们发现TNFALPHA激活其他转录因子IRF-1，STAT1和STAT2，并差异地调节CD38，IL-6，RANTES和EOTAXIN的表达，而不是通过分泌的IFNBETA的自动分泌作用来调节ICAM-1。因此，该竞争性延续建议的目的旨在通过确定这些信号分子在调节TNFalpha对合成功能的调节中的相对贡献来扩展我们以前的发现。该提案的中心假设指出，TNFALPHA和自分泌IFNBETA在协同作用中作用于STAT1/STAT1/STAT2，NF-KAPPAB和CD38依赖性途径的协同调节炎症基因表达。在AIM 1中，我们将确定NF-kappab是否与STAT1/STAT2和IRF-1功能相互作用，以使用细胞因子启动子构建体以及共免疫沉淀，共钙化和GEL移位分析以IFNBETA依赖性方式调节TNFalpha诱导的炎症基因的表达。在AIM 2中，我们将使用主要的负活性cDNA构建体来确定调节人ASM细胞中细胞因子诱导的IFNBETA表达的转录机制（TRAFIKK）。我们还将通过表征ASM中IFNBETA信号分子的体内表达和/或激活IFNBETA信号分子的时间过程，从而在ASM中通过ASM在ASM中确定IFNBETA表达的体内相关性。在AIM 3中，我们将表征IFNBETA依赖性CD38途径是否需要和/或足以调节TNFalpha诱导的ASM细胞中炎症基因的表达。 CD38的作用将使用CD38 - / - ASM和可溶性抑制剂进行研究。依赖钙依赖性途径在TNFalpha诱导基因中的作用将通过使用基因缺陷型小鼠，报告基因构建体和可溶性抑制剂来检查FKBP12.6和NF-AT的作用。由于炎性细胞因子/趋化因子在哮喘中的重要性，我们的研究结果将确定ASM衍生的细胞因子不仅通过旁分泌作用，而且通过ASM合成功能的自分泌调节来调节气道炎症的调节。因此，了解调节ASM炎症基因表达的转录机制可能会导致新的治疗哮喘治疗方法。