Correcting Pathogenic TGF beta Activity in the Airway

摘要 肺内呼吸道表面液体(ASL)的动态平衡严重依赖于氯离子(Cl-)的转运， 由根尖通道CFTR(囊性纤维化跨膜电导调节因子)介导。Cftr基因 与高加索人最常见的常染色体隐性遗传病-囊性纤维化相关的突变 导致CFTR功能丧失，ASL容量调节严重受损。删除F508 CFTR基因(F508del)在90%的CF患者中存在；它阻止CFTR的生物合成过程，减少 CFTRCl-通道功能减弱，降低了CFTRmRNA的稳定性和转移性。据估计，经典的 当CFTR通道功能低于正常的1%和至少10%时，会出现严重的CF表型 需要功能来缓解严重的表型。针对CFs基本分子缺陷的治疗 显示出潜力，但对大多数患者来说还不够。而FDA批准的药物VX-770增强了 CFTR通道“开放”的概率和改善肺功能的不到10%的罕见患者 突变G551D，结合使用VX-770和校正器VX-809，挽救折叠和加工 缺陷对F508del患者只有轻微的效果。Cf肺部疾病较严重的患者 对这种疗法完全抗拒。转化生长因子-β-1参与矫正器的抵抗 阻断CFTR翻译和抑制对ASL残余稳态至关重要的辅助离子通道的治疗 在CF中，即BK(钙激活的K+通道)和ANO1(钙激活的氯离子通道)。高转化生长因子-β-1水平 在40%的F508del纯合子患者中，由于转化生长因子-β1基因的多态而出现这种情况。空气污染物， 包括香烟烟雾也会增加转化生长因子-β-1水平。相伴而生的转化生长因子-β-1的上调 增加F508del纯合子患者的CF肺部疾病的严重程度，并对 针对F508del-CFTR救援的治疗。我们认为，靶向致病转化生长因子-β1活性将 通过恢复BK和ANO1通道的功能，改善CFASL残余容量的动态平衡。它 还将通过恢复减少的508del-CFTR来提高对F508del CF患者的治疗效果 翻译并允许突变的CFTR通道功能的活性增加。因此，定义转化生长因子-β-1 肺组织中的介质为设计消除致病转化生长因子-β1的新药提供了机会 慢性萎缩性胃炎患者的活动性。我们的中心假设是，定向降低转化生长因子-β1的活性将改善 对CFTR、BK和ANO1通道和ASL动态平衡的负面影响，并提供了一种新的方法 治疗F508del CF患者。我们的具体目标是检验假设：(1)DAB2是一个转化生长因子-β1适配器， 通过引导Smad3的核转运抑制纤维支气管壁上皮残留的ASL动态平衡 抑制辅助通道BK和ANO1；(2)DAB2-Smad3相互作用上调microRNAs 这抑制了辅助通道并阻断了校正器介导的F508del-CFTR的救援；以及(3) 转化生长因子-β-1的致病活性需要DAB_2-Smad3相互作用。

ABSTRACT Homeostasis of the airway surface liquid (ASL) in lung is critically dependent on chloride ion (Cl-) transport, mediated by the apical channel, CFTR (cystic fibrosis transmembrane conductance regulator). CFTR gene mutations associated with the most common autosomal recessive disease in Caucasians, cystic fibrosis (CF) result in loss of CFTR function and severe impairment of the ASL volume regulation. Deletion of F508 (F508del) in the CFTR gene is present in 90% of CF patients; it blocks CFTR biosynthetic processing, reduces CFTR Cl- channel function, and decreases CFTR mRNA stability and translation. It is estimated that the classic severe CF phenotype develops when the CFTR channel function is less than 1% of normal and at least 10% function is needed to alleviate the severe phenotype. Therapies targeting the basic molecular defects in CF demonstrate potential but are insufficient for most patients. While the FDA-approved drug VX-770 potentiates CFTR channel “open” probabilities and improves lung function for less than 10% of patients with the rare mutation G551D, the combined use of VX-770 and a corrector VX-809 that rescues the folding and processing defect was only marginally effective for F508del patients. CF patients with more severe lung disease were entirely resistant to such therapy. Transforming growth factor (TGF)-β1 contributes to resistance of corrector therapy by blocking CFTR translation and represses ancillary ion channels critical to residual ASL homeostasis in CF, namely BK (a Ca+2-activated K+ channel) and ANO1 (a Ca+2-activated Cl- channel). High TGF-β1 levels are seen in 40% of F508del homozygous patients due to polymorphisms in the TGF-β1 gene. Air pollutants, including cigarette smoke also increase TGF-β1 levels. The concomitant upregulation of TGF-β1 in turn increases the severity of CF lung disease in F508del homozygous patients and presents a major block to therapies aimed at F508del-CFTR rescue. We propose that targeting the pathogenic TGF-β1 activity would improve the residual ASL volume homeostasis in CF by restoring the function of BK and ANO1 channels. It would also increase the efficacy of therapy for F508del CF patients by restoring the diminished 508del-CFTR translation and allowing increased activity of the mutant CFTR channel function. Thus, defining TGF-β1 mediators in lung tissue presents an opportunity to design novel drugs eliminating the pathogenic TGF-β1 activity in CF patients. Our central hypothesis is that the targeted reduction of TGF-β1 activity will ameliorate negative effects on CFTR, BK, and ANO1 channels and ASL homeostasis, and provide a novel approach to treat F508del CF patients. Our specific aims are to test the hypothesis: (1) that Dab2 is a TGF-β1 adaptor that inhibits the residual ASL homeostasis in CF bronchial epithelium by directing nuclear transport of Smad3 to repress the ancillary channels, BK and ANO1; (2) that the Dab2-Smad3 interactions upregulates microRNAs that repress the ancillary channels and block corrector-mediated rescue of F508del-CFTR; and (3) that a Dab2-Smad3 interaction is required for the pathogenic activity of TGF-β1 in CF bronchial epithelium.