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/或逆转慢性支气管炎药物的策略是 实验性地剖析了烟草烟雾与粘蛋白过度产生之间的信号通路， 肺上皮细胞为了最快地移动，我们最初选择使用以下方法建立疾病模型： 同源细胞系最便于蛋白质和RNA分析。以这样 实验中，我们发现烟雾会引发氧自由基（活性氧）的产生 物种，ROS）通过NADPH氧化酶。ROS反过来刺激肿瘤坏死因子α 转化酶（TACE，ADAM 17金属蛋白酶）切割跨膜 双调蛋白是表皮生长因子受体（EGFR）的配体。的结合 双调蛋白与EGFR结合，然后刺激受体信号传导，导致MAP激活 激酶erk 1/2和信号通路最终激活AP-1反应元件 位于MUC 5AC转录起始位点上游约3.5kb处。虽然这些结果 暂时提出了某些药物靶点，结果应该在更多的生理学验证 相关制度先行。此外，细胞系的初步工作留下了一些问题， 悬而未决其中一个问题是，哪些机制介导EGFR非依赖性 粘蛋白诱导基于该提案中显示的证据，我们假设钙激活的CI通道CLCA 1在这些机制中起作用。为了了解CLCA 1和MUC 5AC诱导之间的关系，我们将进行通道的定点突变，以检测蛋白质-蛋白质相互作用的潜在位点。为了寻找CLCA 1的信号传导伙伴，我们将对从烟雾暴露的组织或细胞中产生的cDNA文库进行酵母2杂交筛选。这些研究将验证和扩展早期的数据粘蛋白诱导的烟雾机制。