Role of Calcium-Activated Chloride Channels in Airway Disease Phenotypes

气道高反应性和黏液细胞化生是炎症性呼吸道的基本特征 疾病(包括哮喘和COPD)，但导致这些疾病的细胞和分子途径 特征仍然需要更好地定义。其他人之前的研究表明，高脚杯细胞特有的成员 钙激活氯离子通道蛋白家族(小鼠的mClca3和人的hCLCA1)的 过敏原诱导的气道高反应性和杯状细胞化生的充要条件 并在人类过敏性哮喘中过度表达。但是，我们发现一个新的/NC/CA3-NULL 在病毒感染或过敏原攻击后，小鼠仍然完全发展为这些哮喘特征。这些和其他 初步结果表明，CLCA基因家族存在功能冗余。事实上，我们发现《中医药学公约》 基因座由老鼠的6个不同的基因和人类的4个不同的基因组成，每个基因都表现出高度的 物种内和物种间的序列同源性，但在组织中的表达模式截然不同。因此，我们 建议选定的CLCA蛋白家族成员在组织特异性但趋同的 气道高反应性和杯状细胞化生的发生和维持。因此，我们 建议： I.在实验环境中确定mClca家族成员的基因表达模式 用实时定量RT-PCR检测病毒感染或变应原刺激引起的呼吸道疾病 精确监测野生型和mClca3缺失型MC/ca家族基因表达水平的方法 老鼠。将使用我们的病毒性毛细支气管炎和过敏原的小鼠模型在体内进行研究 气液界面原代培养的小鼠呼吸道上皮细胞的体外培养 并用细胞因子(IL-4、IL-9、IL-13)刺激诱导杯状细胞化生。 确定选择性表达mClca家族成员在实验性呼吸道疾病中的作用 使用腺相关技术的表型(如杯状细胞化生和呼吸道高反应性) 病毒(AAV)基因转移载体。体内研究将再次与mclca的作用进行比较。 使用原代培养的小鼠和/或人类呼吸道上皮细胞在体外进行表达。 确定选择性阻断mClca家族成员的表达对实验动物的影响 病毒感染或变应原挑战导致的呼吸道疾病表型，使用AAV载体传递 选择性抑制mClca基因表达的反义寡核苷酸。这将与 靶向突变(即，我们的MC/CA3基因缺失小鼠)和丹尼氟酸酯(AN)治疗的效果 依赖钙的氯离子通量的抑制剂)。活体研究将再次与阿司匹林 这些抑制剂在体外使用原代培养的小鼠和/或人类呼吸道上皮细胞。 这些拟议的实验应该用来确定实验性哮喘的CLCA功能，从而 为患有呼吸道疾病的人类提供新的治疗靶点。

Airway hyperresponsiveness and mucous cell metaplasia are essential features of inflammatory airway diseases (including asthma and COPD), but the cellular and molecular pathways leading to these disease traits still need to be better defined. Previous work by others suggested that a goblet cell-specific member of the calcium activated chloride channel family of proteins (mClca3 in mouse and hCLCAl in human) is necessary and sufficient for allergen-induced airway hyperresponsiveness and goblet cell metaplasia in mice and is overexpresssed in allergic asthma in humans. However, we found that a new /nC/ca3-null mouse still fully develops these asthma traits after viral infection or allergen challenge. These and other preliminary results suggest functional redundancy in the CLCA gene family. Indeed, we find that the CLCA locus consists of 6 distinct genes in the mouse and 4 in the human, and each exhibits a high degree of intra- and inter-species sequence homology but a distinct pattern of expression in tissues. We therefore propose that select members of the CLCA family of proteins play tissue-specific but convergent roles in the development and maintenance of airway hyperresponsiveness and goblet cell metaplasia. Thus, we propose to: I. Determine the patterns of gene expression for mClca family members in the setting of experimental airway disease driven by viral infection or allergen challenge, using real-time quantitative RT-PCR assays to precisely monitor the levels of mC/ca family gene expression in wild-type and mClca3-null mice. Studies will be performed in vivo using our mouse models of viral bronchiolitis and allergen challenge and in vitro using primary-culture mouse airway epithelial cells grown at air-liquid interface and stimulated with cytokines (IL-4, IL-9, and IL-13) to induce goblet cell metaplasia. II. Determine the effects of selectively expressing mClca family members on experimental airway disease phenotypes (i.e.goblet cell metaplasia; and airway hyperresponsiveness) using adeno-associated virus (AAV) gene transfer vectors. In vivo studies will again be compared to the actions of mClca expression done in vitro using primary-culture mouse and/or human airway epithelial cells. III. Determine the effects of selectively blocking expression of mClca family members on experimental airway disease phenotypes driven by viral infection or allergen challenge, using AAV vectors to deliver antisense oligonucleotides that selectively inhibit mClca gene expression. This will be compared to effects of targeted mutagenesis (i.e., our mC/ca3-null mouse) and of treatment with taniflumate (an inhibitor of calcium-dependent chloride flux). In vivo studies will again be compared to the actions of these inhibitors in vitro using primary-culture mouse and/or human airway epithelial cells. These proposed experiments should serve to define CLCA function in experimental asthma and thereby provide for new therapeutic targets in humans with airway disease.