Role of MU5AC Mucins in Airway Disease

描述(申请人提供)：呼吸道粘液阻塞是囊性纤维化(CF)患者发病和死亡的主要原因，主要归因于粘蛋白糖蛋白(粘蛋白)的过度生产和过度分泌。持续的粘蛋白高分泌需要增加粘蛋白(MUC)基因的表达，该基因编码粘蛋白的蛋白质骨架。最近的报告表明，在细菌感染之前，CF呼吸道具有固有的高炎症环境，促进粘蛋白过度产生。我们最近发现，IL8通过增加粘蛋白mRNA的稳定性，在体外增加了两个呼吸道粘蛋白基因MUC5AC和MUC5B的表达。重要的是，感染前后CF患者呼吸道中IL8水平的升高表明IL8可以在体内启动和维持粘蛋白基因的表达。糖皮质激素通过多因素机制减少体内的呼吸道炎症和粘蛋白的产生，包括改变抗炎蛋白或细胞因子的表达。GC在体外也能降低粘蛋白基因的表达。更好地了解促炎和抗炎介质对粘蛋白基因的调控，以及CF肺中炎症介质的个体发育，最终应该会导致绕过CF气道粘蛋白基因表达增加和粘液阻塞的治疗。为实现这一目标，提出了三个目标。在目标1中，IL8通过改变识别粘蛋白基因3‘非翻译区特定顺式序列的RNA结合蛋白(RBP)的表达或通过介导调节或编码RBP的基因的表达来增加呼吸道上皮细胞中粘蛋白mRNA的稳定性的假说将通过EMSA、突变分析、基因表达分析和基因表达分析来验证。在目标2中，将研究GC通过下调与MUC5AC 5‘上游侧翼序列中GC反应元件相互作用的转录因子和/或通过改变介导粘蛋白基因表达的抗炎或促炎基因的表达来降低MUC5AC基因在呼吸道上皮细胞中的表达。在目标3中，将通过对慢性阻塞性肺疾病患者和非慢性阻塞性肺疾病患者呼吸道组织的基因表达谱分析来识别和评估与呼吸道粘蛋白过度产生有关的候选基因，以验证这样的假设，即慢性阻塞性肺疾病患者的气道粘液阻塞是有缺陷的CF基因的结果，这会导致调节呼吸道内稳的基因的表达发生变化，包括粘蛋白的产生。

DESCRIPTION (provided by applicant): Airway mucus obstruction, a major cause of morbidity and mortality in cystic fibrosis (CF) patients, is largely attributed to overproduction and hypersecretion of mucin glycoproteins (mucins). Sustained mucin hypersecretion requires increased expression of mucin (MUC) genes that encode the protein backbone of mucins. Recent reports have indicated that CF airways have an intrinsically high inflammatory milieu, prior to bacterial infection, that promotes mucin overproduction. We have recently shown that IL8 increases in vitro expression of two airway mucin genes, MUC5AC and MUC5B, by increasing mucin mRNA stability. Importantly, elevated levels of IL8 in the airways of CF patients prior to and after infection suggest that IL8 can initiate and maintain increased mucin gene expression in vivo. Glucocorticoids decrease airway inflammation and mucin production in vivo by multi-factorial mechanisms, including alterations in expression of anti-inflammatory proteins or cytokines. GC also decreases mucin gene expression in vitro. A better understanding of mucin gene regulation by pro-and anti-inflammatory mediators, as well as the ontogeny of inflammatory mediators in CF lungs, should ultimately result in therapies that circumvent increased mucin gene expression and mucus obstruction in CF airways. Three aims are proposed to address this objective. In Aim 1, the hypothesis that IL8 increases mucin mRNA stability in airway epithelial cells by altering expression of RNA-binding proteins (RBP) that recognize specific cis-sequences in the 3' untranslated region of mucin genes or by mediating expressions of genes that regulate or encode RBP will be tested by EMSA, mutation analysis, transfection assays and gene expression analyses. In Aim 2, the hypothesis that GC decrease MUC5AC gene expression in airway epithelial cells by down-regulating transcriptions factors that interact at or near GC response elements in the 5'-upstream flanking sequences of MUC5AC and/or by altering expression of anti-or pro-inflammatory genes that mediate mucin gene expression will be investigated. In Aim 3, candidate genes involved in airway mucin overproduction will be identified and assessed by gene expression array profiling of respiratory tract tissues from CF and non-CF patients to test the hypothesis that airway mucus obstruction in CF patients is a consequence of the defective CF gene, which results in altered expression of genes that mediate airway homeostasis, including mucin production.