Role of Chloride Channels in Mucin Production

氯离子通道在粘蛋白生产中的作用

• 批准号: 6723666
• 负责人: CAROL B BASBAUM
• 金额: $ 37.88万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6723666
• 关键词: biological signal transduction; biopsy; bronchoscopy; chloride channels; clinical research; disease /disorder model; enzyme inhibitors; epidermal growth factor; free radical oxygen; free radical scavengers; gene induction /repression; glycoprotein biosynthesis; growth factor receptors; human subject; laser capture microdissection; metalloendopeptidases; model design /development; molecular film; mucins; protein protein interaction; protein structure function; respiratory epithelium; site directed mutagenesis; smoking; tobacco; yeast two hybrid system

DESCRIPTION (provided by applicant): Our strategy for the development of drugs to prevent and/or reverse chronic bronchitis is to experimentally dissect signaling pathways linking tobacco smoke to mucin overproduction in lung epithelial cells. To move most rapidly, we initially chose to establish disease models using homogeneous cell lines that were most convenient for protein and RNA analysis. In such experiments, we found that smoke triggers the generation of oxygen radicals (reactive oxygen species, ROS) by NADPH oxidase. The ROS, in turn, stimulate tumor necrosis factor alpha converting enzyme (TACE, ADAM 17 metalloproteinase) to cleave transmembrane amphiregulin, a ligand for the epidermal growth factor receptor (EGFR). The binding of amphiregulin to EGFR then stimulates receptor signaling resulting in the activation of the MAP kinase erk 1/2 and the signaling pathway culminates in activation of an AP-1 response element located about 3.5 kb upstream of the MUC 5 AC transcription start site. Although these results tentatively suggest certain drug targets, the results should be validated in more physiologically relevant systems prior to proceeding. In addition, the initial work in cell lines left certain issues unresolved. Among these is the question of which mechanisms mediate EGFR-independent mucin induction. Based on evidence shown in the proposal, we hypothesize that the calciumactivated CI channel, CLCA1 plays a role in such mechanisms. To understand the relationship between CLCA1 and MUC 5AC induction by smoke, we will perform site-directed mutagenesis of the channel to detect potential sites of protein-protein interaction. To pursue signaling partners of CLCA1, we will perform yeast 2 hybrid screens of cDNA libraries created from smoke- exposed tissue or cells. These studies will validate and extend earlier data concerning the mechanism of mucin induction by smoke.

描述（由申请人提供）： 我们开发预防和/或反向慢性支气管炎的药物的策略是 实验剖析将烟草烟与粘蛋白过量生产联系起来的信号通路 肺上皮细胞。为了最快地移动，我们最初选择使用 对于蛋白质和RNA分析最方便的均匀细胞系。在这样 实验，我们发现烟雾会触发氧自由基的产生（活性氧 NADPH氧化酶的物种，ROS）。 ROS反过来刺激肿瘤坏死因子α 将酶（TACE，ADAM 17金属蛋白酶）转换为裂解跨膜 两极蛋白，表皮生长因子受体（EGFR）的配体。结合 然后，两次凝血蛋白到EGFR，然后刺激受体信号传导，导致地图激活 激酶ERK 1/2和信号通路最终在AP-1响应元件的激活中达到顶点 位于MUC 5 AC转录起始站点上游约3.5 kb。虽然这些结果 暂时提出某些药物靶标，应在生理上更验证结果 在进行之前，相关系统。此外，细胞系中的最初工作留下了某些问题 未解决。其中包括哪些机制介导了EGFR无关的问题 粘蛋白诱导。根据提案中显示的证据，我们假设钙活化的CI通道CLCA1在此类机制中起作用。为了了解CLCA1和MUC 5AC通过烟雾诱导的关系，我们将执行通道的位置定向诱变，以检测蛋白质 - 蛋白质相互作用的潜在位点。为了追求CLCA1的信号伴侣，我们将执行由烟雾暴露的组织或细胞产生的cDNA文库的酵母2混合筛选。这些研究将验证和扩展有关烟雾诱导粘蛋白机理的早期数据。