Role of Calcium-Activated Chloride Channels in Airway Disease Phenotypes

气道高反应性和粘液细胞化生是炎症气道的基本特征 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。其他人先前的工作表明，一个杯状细胞特异性成员 钙激活的氯离子通道蛋白家族（小鼠中的mClca 3和人中的hCLCA 1）中的一个是 在过敏原诱导的气道高反应性和杯状细胞化生中， 在小鼠中表达，并在人类过敏性哮喘中过表达。然而，我们发现一个新的/nC/ca 3-null 小鼠在病毒感染或过敏原攻击后仍然完全发展这些哮喘特征。这些和其他 初步结果提示CLCA基因家族中存在功能冗余。事实上，我们发现， 该基因座由小鼠中的6个不同基因和人类中的4个不同基因组成，并且每个基因都表现出高度的特异性。 种内和种间序列同源性，但组织中的表达模式不同。因此我们 提出CLCA家族蛋白质的选择成员在组织中发挥组织特异性但收敛的作用， 发展和维持气道高反应性和杯状细胞化生。因此我们 提议： I.在实验环境中确定mClca家族成员的基因表达模式， 使用实时定量RT-PCR检测由病毒感染或过敏原激发引起的气道疾病 精确监测野生型和mClca 3-null中mC/ca家族基因表达水平的测定 小鼠将使用我们的病毒性细支气管炎和过敏原小鼠模型进行体内研究。 激发和体外使用在气-液界面生长的原代培养小鼠气道上皮细胞 并用细胞因子（IL-4、IL-9和IL-13）刺激以诱导杯状细胞化生。 二.确定选择性表达mClca家族成员对实验性气道疾病的影响 表型（即杯状细胞化生;和气道高反应性）使用腺相关 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/或人气道上皮细胞在体外进行表达。 三.确定选择性阻断mClca家族成员的表达对实验性 由病毒感染或过敏原攻击驱动的气道疾病表型，使用AAV载体递送 选择性抑制mClca基因表达的反义寡核苷酸。这将与 靶向诱变的效果（即，我们的mC/ca 3-null小鼠）和用他尼福酯（一种 钙依赖性氯化物通量抑制剂）。体内研究将再次与以下的作用进行比较： 这些抑制剂在体外使用原代培养的小鼠和/或人气道上皮细胞。 这些拟议的实验应有助于确定CLCA在实验性哮喘中的功能， 为患有呼吸道疾病的人提供新的治疗靶点。

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.