Role of Chloride Channels in Mucin Production

描述(由申请人提供)： 我们开发预防和/或逆转慢性支气管炎的药物的战略是 在实验中剖析烟草烟雾与粘蛋白过度产生之间的信号通路 肺上皮细胞。为了最快地推进，我们最初选择使用以下方法建立疾病模型 最便于蛋白质和RNA分析的同源细胞系。在这样的情况下 实验中，我们发现烟雾会触发氧自由基(活性氧)的产生 物种，ROS)的NADPH氧化酶。ROS反过来刺激肿瘤坏死因子α 转换酶(TACE、ADAM 17金属蛋白酶)切割跨膜 双调蛋白，表皮生长因子受体(EGFR)的配体。的约束性 然后，EGFR的双调节素刺激受体信号，导致MAP的激活 激酶ERK1/2和信号通路最终激活AP-1反应元件 位于MUC 5 AC转录起始点上游约3.5kb。尽管这些结果 试探性地建议某些药物靶点，结果应该在更多的生理上得到验证 在继续进行之前，应建立相关制度。此外，最初在细胞系中的工作留下了一些问题 悬而未决。其中一个问题是，哪些机制调节了EGFR的非依赖性 粘蛋白诱导。根据提案中显示的证据，我们假设钙激活的CI通道CLCA1在这种机制中发挥作用。为了了解CLCA1与烟雾诱导MUC 5AC之间的关系，我们将对该通道进行定点突变，以检测潜在的蛋白质-蛋白质相互作用位点。为了寻找CLCA1的信号伙伴，我们将对烟雾暴露的组织或细胞中创建的cDNA文库进行酵母2杂交筛选。这些研究将验证和扩展有关烟雾诱导粘蛋白机制的早期数据。

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.