ROLE OF CFTR IN PROCESSING AND FUNCTION OF MUC1 MUCIN

CFTR 在 MUC1 粘蛋白加工和功能中的作用

• 批准号: 6499600
• 负责人: joseph pilewsky
• 金额: $ 12.41万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6499600
• 关键词: Pseudomonas aeruginosa; antisense nucleic acid; bacteria infection mechanism; cell adhesion; chloride channels; cyclic AMP; cystic fibrosis; flow cytometry; gene mutation; human tissue; immunocytochemistry; immunoelectron microscopy; intracellular transport; molecular pathology; mucins; posttranslational modifications; protein localization; protein structure function; protein transport; respiratory epithelium; sulfation; tissue /cell culture

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.

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。MUC1黏液蛋白是人类肺中唯一已知的跨膜黏液蛋白，在分化的表面上皮细胞和粘膜下腺的浆液细胞中表达，这些细胞先前已被证明表达高水平的囊性纤维化跨膜传导调节因子（CFTR）。MUC1和CFTR的细胞共定位，以及CFTR在糖缀合物翻译后加工中的作用，表明CFTR突变可能改变MUC1的加工。此外，MUC1的结构和空间分布与气道上皮顶膜与腔内细菌的相互作用有关。因此，本研究的中心假设是，CF突变损害了MUC1的翻译后加工和释放，从而损害了MUC1对正常气道防御细菌感染的作用。因此，具体目标是：1。目的：明确跨膜粘蛋白MUC1在正常和病变人气道上皮及分泌物中的细胞定位。MUC1在人气道和临床相关的原发CF和非CF人气道上皮体外系统中的表达和细胞分布将通过免疫细胞化学、流式细胞术和免疫电镜来确定。2. 确定CFTR是否影响MUC1的翻译后加工。MUC1的硫酸化和唾液化将在CALU-3细胞以及CF和非CF上皮的原代培养中测定。在CALU-3细胞中使用反义方法下调CFTR的表达。3. 确定MUC1在CF气道上皮中是否异常加工和释放。在非CF和CF气道上皮中，存在和不存在cAMP刺激时MUC1运输和释放的动力学将被确定。在cAMP刺激存在和不存在的情况下，MUC1运输和释放的动力学将在非CF和CF气道上皮以及表达不同水平CFTR的CALU-3细胞中确定。4. 目的探讨CF突变对MUC1与铜绿假单胞菌相互作用的影响。将比较原代气道细胞和CFTR和/或MUC1表达下调的CALU-3细胞中的细菌粘附。这些目标的完成将明确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。