ROLE OF CFTR IN PROCESSING AND FUNCTION OF MUC1 MUCIN

在确定CF基因突变导致气道感染和支气管扩张的机制方面已经取得了重大进展，但异常粘蛋白加工在CF肺病发病机制中的潜在作用仍不清楚。粘蛋白MUC 1是人类肺中唯一已知的跨膜粘蛋白，并且在分化的表面上皮细胞和粘膜下腺体的浆液细胞中表达，其先前已贝内证明表达高水平的囊性纤维化跨膜传导调节因子（CFTR）。MUC 1和CFTR的细胞共定位，以及CFTR在糖缀合物的翻译后加工中的报道作用，表明CFTR突变可能改变MUC 1的加工。此外，MUC 1的结构和空间分布暗示其参与气道上皮细胞顶膜与管腔细菌之间的相互作用。因此，该提议的中心假设是CF突变损害MUC 1的翻译后加工和释放，并且这损害MUC 1对正常气道防御细菌感染的贡献。因此，具体目标是：1。确定跨膜粘蛋白MUC 1在正常和患病人气道上皮和分泌物中的细胞定位。将使用免疫细胞化学、流式细胞术和免疫电子显微镜来确定MUC 1在人气道中以及在原发性CF和非CF人气道上皮的临床相关体外系统中的表达和细胞分布。2.确定CFTR是否影响MUC 1的翻译后加工。将在CALU-3细胞以及CF和非CF上皮细胞的原代培养物中测定MUC 1的硫酸化和唾液酸化。在CALU-3细胞中使用反义方法下调CFTR表达。3.目的：探讨粘蛋白1（MUC 1）在CF气道上皮细胞中的异常加工和释放。将在非CF和CF气道上皮中测定在存在和不存在cAMP刺激的情况下MUC 1运输和释放的动力学。在存在和不存在cAMP刺激的情况下，MUC 1运输和释放的动力学将在非CF和CF气道上皮中以及在表达各种水平的CFTR的CALU-3细胞中确定。4.确定CF突变对MUC 1和铜绿假单胞菌之间相互作用的影响。将在原代气道细胞和其中CFTR和/或MUC 1表达已下调的CALU-3细胞中比较细菌粘附。这些目标的完成将确定CFTR对人类气道中特定粘蛋白的加工和功能的影响，从而提高对CF气道感染发病机制的理解。

Significant progress has been made towards defining the mechanisms whereby mutations in the CF gene lead to airway infection and bronchiectasis, but the potential role of aberrant mucin processing in the pathogenesis of CF lung disease remains unclear. The mucin MUC1 is the only known transmembrane mucin the human lung, and is expressed in differentiated surface epithelial cells and in the serous cells of submucosal glands, which previously have bene shown to express high levels of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The cellular co-localization of MUC1 and CFTR, and the reported role of CFTR in the post-translational processing of glycoconjugates, suggest that CFTR mutations may alter the processing of MUC1. Moreover, the structure and spatial distribution of MUC1 implicate it in the interaction between the apical membrane of airway epithelia and luminal bacteria. The central hypotheses of this proposal re therefore that CF mutations impair the post- translational processing and release of MUC1, and that this impairs the contribution of MUC1 to normal airway defense against bacterial infection. Accordingly, specific aims are: 1. To define the cellular localization of the transmembrane mucin MUC1 in normal and diseased human airway epithelium and secretions. The expression and cellular distribution of MUC1 in human airway and in a clinically relevant in vitro system of primary CF and non-CF human airway epithelium will be determined using immunocytochemistry, flow cytometry, and immunoelectron microscopy. 2. To determine whether CFTR influences the post-translational processing of MUC1. The sulfation and sialylation of MUC1 will be determined in CALU-3 cells and in primary cultures of CF and non-CF epithelia. CFTR expression will be down regulated using an anti-sense approach in CALU-3 cells. 3. To determine whether MUC1 is aberrantly processed and released in CF airway epithelia. The kinetics of MUC1 trafficking and release in the presence and absence of cAMP stimulation will be determined in non-CF and CF airway epithelia. The kinetics of MUC1 trafficking and release in the presence and absence of cAMP stimulation will be determined in non- CF and CF airway epithelia, and in CALU-3 cells expressing various levels of CFTR. 4. To determine the impact of CF mutations in the interaction between MUC1 and Pseudomonas aeruginosa.. Bacterial adhesion will be compare din primary airway cells, and in CALU-3 cells in which CFTR and/or MUC1 expression have been down regulated. Completion of these aims will define the influence of CFTR on the processing and function of a specific mucin in human airway and thereby improve the understanding of the pathogenesis of airway infection in CF.